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PubMed1989

Image reconstruction of the flagellar basal body of Salmonella typhimurium.

The basal body is thought to be a part of the rotary motor of the bacterial flagellum. It consists of a central rod coaxial with a set of four rings, which are associated with the cell envelope. We used single-particle averaging methods to analyze images of negatively stained basal bodies of Salmonella typhimurium. Several averages were computed, so that the reliability of features could be assessed. We carried out the same analysis on electron micrographs of isolated, negatively stained L-P rings. In order to interpret the averages in terms of a three-dimensional structure, we carried out image reconstruction on them. The resulting three-dimensional map corresponds to the cylindrically averaged structure of the basal body. To show that the reconstruction procedure is legitimate, we demonstrate it on model data. The results of the modelling show that features very near to the axis of the reconstruction are not reliable but that broader, off-axis features are represented faithfully. The L ring is L-shaped, with the long arm of the L parallel to the axis of the rod, and the short arm pointing away from the rod. The P ring, on the other hand, appears to be a ring or disk. The position of the L-P ring complex on the rod seems to vary somewhat, consistent with its putative role as a bushing. The cross-sectional shape of the S ring is that of a frustum rather than a disk. The M ring, which is oval in cross section, sits atop the S ring, making contact with it at an outer radius. The S ring appears to make contact with the rod, whereas the M ring does not. This situation, if true, is difficult to reconcile with the common notion that the S ring is stationary and the M ring rotates. It seems more likely that the S ring and rod rotate as a unit.

Flagella↗

PubMedDate not supplied

Visualization of myosin helices in sections of rapidly frozen relaxed tarantula muscle.

Tarantula leg muscles in the relaxed state were rapidly frozen against a copper block cooled with liquid helium. Thin longitudinal sections of freeze-substituted specimens, both live and skinned, clearly showed the helical tracks of crossbridges on the surface of the myosin filaments, which are not preserved by conventional fixation. Fourier transforms of selected filaments showed a myosin layer line pattern, similar to that observed in X-ray diffraction patterns of intact tarantula muscle, extending to the sixth order of the 43.5 nm X-ray repeat. The phases of corresponding reflections were similar on the two sides of the meridian on the first layer line, and the crossbridge arrangement showed a line of mirror symmetry running down the center of the filament. These observations show that the number of helices (N) is even, in agreement with N = 4 determined from image analysis of negatively stained, isolated tarantula filaments (Crowther et al., J. Mol. Biol. 184, 429-439, 1985). Filtered images showed clear detail of the crossbridge helices and were similar to filtered images of negatively stained, isolated thick filaments. Thus, rapid freezing combined with freeze-substitution preserves the crossbridges in a three-dimensional arrangement approximating that occurring in vivo.

Animals↗

PubMed1994

Characterization of the chloroplast cytochrome b6f complex as a structural and functional dimer.

Size analysis of the cytochrome b6f complex by FPLC Superose-12 chromatography and Blue Native PAGE indicated a predominantly dimeric component with M(r) = (1.9-2.5) x 10(5). The true dimer molecular weight including bound lipid, but not detergent, was estimated to be 2.3 x 10(5). Size and shape analysis by negative-stain single-particle electron microscopy indicated that the preparation of dimeric complexes contains a major population that has a protein cross section 40% larger than the monomer, binds more negative stain, and has a geometry with a distinct 2-fold axis of symmetry compared to the monomeric complex. The dimeric species is more stable at higher ionic strength with respect to conversion to the monomeric species. SDS-PAGE of monomer and dimer preparations indicated that both contain the four major polypeptides in approximately equal stoichiometry and also contain the petG M(r) 4000 subunit. One bound chlorophyll a per monomer, part of the bound lipid, is present in monomer and dimer. The in vitro electron-transport activity (decyl-PQH2-->PC-ferricyanide) of the separated dimer was comparable to that of the isolated b6f complex and was 4-5-fold greater than that of the monomer preparation, whose activity could be attributed to residual dimer. No difference in the properties of the dimer and monomer was detected by SDS-PAGE or redox difference spectrophotometry that could account for the difference in activities. However, the concentration of the Rieske [2Fe-2S] center was found by EPR analysis of the gy = 1.90 signal to be lower in the monomer fraction by a factor of 3.5 relative to the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Biopolymers↗

PubMed1984

Structural organization of (Na+ + K+)-ATPase in purified membranes.

The structural organization of crystalline, membrane-bound (Na+ + K+)-ATPase was studied by negative staining and thin sectioning. The enzyme molecules were induced to form crystalline arrays within fragments of membrane by incubation in defined ionic conditions. The enzyme remained fully active after crystallization. Negative staining and computer processing of images of the crystalline specimens identified two discrete crystalline arrays. The dimensions of the unit cell of one of the arrays were large enough to accommodate an alpha beta protomer; those of the other array, an (alpha beta)2 diprotomer . Thin sections of the crystalline fraction contained a unique membrane complex that was formed from two apposed plasma membranes. The paired membranes in this complex were separated by a center-to-center space of 15 nm containing evenly spaced septa that connected the membrane surfaces; the overall thickness of the entire structure was 22-25 nm. The agglutinin from Ricinus communis, a lectin that binds to the carbohydrate moiety of the beta-subunit of (Na+ + K+)-ATPase, decorated the free surfaces of the complex. Therefore, this complex of paired membranes is the result of interactions between the cytoplasmic domains of the enzyme. From measurements of the dimensions of these structures, we estimate the overall length of the enzyme to be approximately 11.5 nm along the axis perpendicular to the plane of the membrane, and the molecular protrudes more (approximately 5 nm) on the cytoplasmic surface than on the extracytoplasmic surface (approximately 2 nm).

Animals↗

PubMed2002

Serous oligocystic adenoma of the pancreas: a clinicopathological and immunohistochemical study of three cases with ultrastructural findings.

AIMS: Serous oligocystic adenoma of the pancreas is an uncommon benign neoplasm and is a recently described entity. To date, there are 19 adult cases of this tumour. We report three additional cases, two with macrocystic and one with unilocular types. We describe their clinicopathological, immunohistochemical and ultrastructural findings and review the world's literature. METHODS: For a 10-year period, we reviewed all benign cystic lesions of the pancreas with emphasis on serous oligocystic adenoma. We characterised serous oligocystic adenoma as an ill-demarcated or encapsulated mass, composed largely or exclusively of macrocysts (cysts measuring 20mm or more) but few in number (oligolocular). Grossly, it may contain only a single cyst (unilocular) of any size with a few satellite cysts observed on histological examination. Special stains and immunohistochemistry as well as electron microscopy were performed on three and two cases of serous oligocystic adenoma, respectively. RESULTS: Between 1990 and 2000, we collected 26 benign cystic lesions of the pancreas, three of which were serous oligocystic adenomas (two with macrocystic and one with unilocular types). Many of the cells lining the cysts showed PAS positivity. There was negative staining for PAS with diastase digestion, Alcian blue and mucicarmine. All cases showed positive staining for CAM5.2, AE1/AE3, EMA and CK7. The proliferation index marker was low. There was negative staining for CK20, insulin, glucagon, somatostatin, synaptophysin, chromogranin A, CEA and p53. Ultrastructural studies on two cases revealed similar findings. The single row of uniform epithelial cells lining the cysts was composed of simple cuboidal to flat cells which rested on a thin basal lamina. Their nuclei were round to ovoid. Glycogen granules were identified in the cytoplasm. Short microvilli emerged from the epithelial apical surface. Adjacent tumour cells were connected by microfilaments. CONCLUSIONS: Serous oligocystic adenomas of the pancreas are uncommon benign tumours. Prior to this study, 19 adults with these lesions were reported in the world's literature. No correct pre-operative diagnosis was carried out on all 22 cases. The 20 patients with follow-up ranging from 2 months to 5 years did not show tumour recurrence or malignant transformation.

Adolescent↗

PubMed1996

Highly ordered two-dimensional crystals of photosystem I reaction center from Synechococcus sp.: functional and structural analyses.

The photosystem 1 reaction center complex from the thermophilic cyanobacterium Synechococcus sp. was isolated by Triton X-100 solubilization and fractional precipitation with polyethylene glycol. As shown by gel electrophoresis, the isolated complex was composed of the 83 kDa subunits A and B, and at least six other subunits with molecular mass below 20 kDa. Electron transfer from the primary electron donor P700 to the FA/FB centers was demonstrated by flash-induced absorption change of the isolated complex, while electron paramagnetic resonance (EPR) spectroscopy showed that the complex contained a full set of Fe-S clusters. Isolated complexes were reconstituted into two-dimensional crystals in the presence of phospholipids and different cations. The crystals were found to be active by flash-induced separation and EPR spectroscopy. Electron microscopy and digital image processing of negatively stained and frozen-hydrated specimens revealed orthorhombic crystals with unit cell dimensions a = 138 A, b = 145 A and p12(1) symmetry. A three-dimensional map was calculated for negatively stained crystals to 19 A resolution, whereas the projection map of frozen-hydrated crystals exhibited 8 A resolution.

Crystallization↗

PubMed1985

Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat.

As the result of a combined biochemical and electron microscopic investigation, hitherto unrecognized structural features of the mouse egg extracellular coat, or zona pellucida, have been revealed. Specimens were prepared for electron microscopy by spraying individually isolated zonae pellucidae onto a substrate and were observed by both rotary shadowing and negative staining techniques. Results of these experiments suggest that the three zona pellucida glycoproteins, ZP1 (200,000 Mr), ZP2 (120,000 Mr) and ZP3 (83,000 Mr), are organized into long filaments. Negatively stained zona pellucida filaments resemble "beads-on-a-string", with each bead (9.5 nm in diameter) located every 17 nm or so (center-to-center distance) along the axis of the filament. The filaments, in turn, appear to be interconnected by one of the three zona pellucida glycoproteins, ZP1, giving rise to a three-dimensional matrix. Proteolysis of ZP1 by chymotrypsin or reduction of intermolecular disulfides of ZP1 by dithiothreitol results in both solubilization of zonae pellucidae and disruption of interconnections between individual zona pellucida filaments. These observations suggest that the zona pellucida, which plays important roles both during and after fertilization of mammalian eggs, is a highly organized extracellular coat in which glycoproteins are assembled into filaments possessing a recognizable structural repeat.

Animals↗

PubMed1979

Electron microscopy of the rodlet layer of Neurospora crassa conidia.

Neurospora crassa macroconidia possess a regularly arranged layer of small fibers (rodlets) near the spore surface. The structure and location of this layer were studied by making surface replicas, by negative staining, by freeze-fracturing and deep-etching, and by thin sectioning. When conidia were shaken vigorously in water, the layer fragmented and became separated from the surface in sheets. Negative staining of such sheets showed that the individual rodlets have a hollow central core. When conidia were shaken gently in water or fixative, large fragments of the rodlet layer often remained on the conidial surface. The fragments tended to fold back on each other such that multiple layers were sometimes seen in thin sections. It is concluded that in dry conidia the rodlets are located on the extreme outside of the spore where they form a monolayer with only occasional regions of overlap.

Cell Wall↗

PubMed1987

Subunit exchange between membranous and soluble forms of bovine dopamine beta-hydroxylase.

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms: soluble and membrane-bound. In a previous study, it was shown that the tetrameric, soluble form of the enzyme undergoes dissociation into two identical dimeric subunits and that this subunit dissociation is dependent on pH and ADP binding (Dhawan, S., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1986) J. Biol. Chem. 261, 7680-7684). Here we report the effect of pH and ADP on the dissociation of the membranous form of dopamine beta-hydroxylase into two nonidentical subunits. Negative stain electron microscopy of purified membranous hydroxylase showed largely tetrameric species together with occasional dimeric species. The tetrameric images of membranous hydroxylase were similar to, but clearly different from, previously published negative stain images of soluble hydroxylase (Duong, L. T., Fleming, P. J., and Ornberg, R. L. (1985) J. Biol. Chem. 260, 2393-2398). Quantitative binding of ADP to the membranous hydroxylase revealed the existence of two binding sites per dimeric subunit. ADP binding and low pH both promote dissociation of a hydrophilic, catalytically active subunit from the membranous enzyme reconstituted onto phospholipid vesicles. Kinetic analyses of reconstituted membranous hydroxylase activity were consistent with the existence of tetrameric and dimeric catalytic species in equilibrium. All of the hydrophilic subunits of the purified soluble hydroxylase bind to the hydrophobic subunits of the reconstituted membranous hydroxylase. We propose that, in the chromaffin granules, the soluble hydroxylase subunits are in equilibrium association with the membrane-bound hydroxylase subunits and that the hydrophilic subunits of both soluble and membranous hydroxylase are identical.

Adenosine Diphosphate↗

PubMed2003

Ovarian tissue banking and fertility preservation in cancer patients: histological and immunohistochemical evaluation.

OBJECTIVE: A combination of chemotherapy and radiotherapy in young females with cancer has greatly enhanced the life expectancy of these patients, even if these treatments have a highly deleterious effect on the ovary and cause a severe depletion of the follicular store. Cryopreservation of ovarian tissue before chemotherapy and/or radiotherapy, followed by autograft after remission or in in vitro maturation, could restore gonadal function and fertility. The aim of this study is to verify the efficiency of the ovarian tissue cryopreservation procedure by histological and immunohistochemical analyses. METHODS: Ovarian tissue was obtained by laparoscopy from 22 patients affected with different malignant diseases. Tissue specimens were frozen using a combination of PROH (1,2-propanediol) and sucrose as cryoprotectants, and the cryopreservation protocol used consisted of a slow freezing-rapid thawing program. Both fresh and frozen/thawed tissues were embedded in paraffin blocks for histological and immunohistochemical analyses. RESULTS: Good stromal and follicular morphology was found in fresh and frozen/thawed tissue. No significant differences were found in follicular density, distribution, and diameters in fresh and frozen/thawed tissue. Follicle immunohistochemical analysis showed a high percentage of negative staining for both estrogen receptor (ER) (100% both in fresh and frozen/thawed specimens) and progesterone receptor (PR) (97% versus 91%, respectively). Regarding the Ki67 protein, positive staining was found in both the granulosa cells and/or the oocytes (36% in fresh and 56% in frozen/thawed). For the Bcl2 protein, positive staining was observed in the follicle granulosa cells but not in the oocytes in 74% of the fresh and in 79% of the frozen/thawed specimens. For the stromal cells, ER showed a negative staining distribution in 97% of the fresh and 100% of the frozen/thawed specimens. The stroma staining distribution was diffuse/focal in fresh versus frozen/thawed specimens (50% versus 74% respectively) for PR, patch/focal (70% versus 80%, respectively) for Ki67 protein, and diffuse (55% versus 54%, respectively) for Bcl2. CONCLUSIONS: These results suggest that human ovarian tissue morphology, antigenicity, cellular proliferation, and anti-apoptotic index were well preserved by cryopreservation in PROH and sucrose.

Adult↗

PubMed1978

The effect of lipopolysaccharide composition on the ultrastructure of Pseudomonas aeruginosa.

The surface structure of Pseudomonas aeruginosa PACl and PAClR and of lipopolysaccharide-defective mutants derived from them was studied by negative-staining and thin-section electron microscopy and compared with that of a rough mutant with wild-type lipopolysaccharide. The rough mutant and the parent strains had fairly smooth outer layers. Negatively stained preparations of all the mutants lacking polymerized O-antigenic sidechains, including a semi-rough mutant, showed numerous blebs on the surface. In thin sections of these mutants occasional extrusions from the surface were seen. They appeared to consist of material extruded from the outer membrane, but there was no evidence to suggest they were complete unit membranes. Polymerized O-antigenic side-chains in the lipopolysaccharide appear to be required to produce the wild-type appearance of the outer membrane in P. aeruginosa.

Antigens, Bacterial↗

PubMed1995

Inability of an isogenic urease-negative mutant stain of Helicobacter mustelae to colonize the ferret stomach.

Eight ferrets specific-pathogen-free for Helicobacter mustelae were given, per dose, approximately 3.0 x 10(7) CFU of either the wild-type parent strain of H. mustelae (NCTC 12032) (two ferrets) the isogenic urease-negative mutant strain of H. mustelae (10::Tn3Km) (four ferrets), or sterile culture broth (two ferrets). Infection status was monitored by endoscopic gastric biopsy for urease activity, histopathology, and culture and by serology at 3, 6, 10, and 21 weeks. All ferrets were necropsied at 25 weeks. Both negative control ferrets remained uninfected, both ferrets receiving the H. mustelae wild-type parent strain became infected after two doses of the organism, and all four ferrets given two doses of the isogenic urease-negative mutant strain of H. mustelae remained uninfected throughout the 6-month study. Histopathology correlated with infection status. H. mustelae-infected ferrets exhibited diffuse mononuclear inflammation in the subglandular region and the lamina propria of the gastric mucosa, while uninfected ferrets showed no or minimal inflammation. These results suggest that urease activity is essential for colonization of the ferret stomach by H. mustelae.

Animals↗

PubMed1989

Scanning transmission electron microscopic study of molluscan hemocyanins in various aggregation states: comparison with light scattering molecular weights.

The masses of individual particles of the hemocyanins of six members of two molluscan classes, Polyplacophora and Gastropoda, have been determined by scanning transmission electron microscopy (STEM) of unstained specimens dried from the frozen state. The decameric hemocyanins of two chitons, Mopalia muscosa and Stenoplax conspicua, had masses of 4.20 +/- 0.18 and 4.47 +/- 0.56 MDa, respectively; the didecameric hemocyanins of two gastropods, Fasciolaria tulipa and Pleuroploca gigantea, had masses of 8.67 +/- 0.44 and 8.96 +/- 0.39 MDa, respectively; and the tridecameric hemocyanin of Lunatia heros had a mass of 13.50 +/- 0.44 MDa. The STEM values were in close agreement with those obtained by light scattering measurements of the same samples in solution. For Busycon contrarium, a gastropod with a multidecameric hemocyanin, nine size classes from didecamers to decadecamers with masses that corresponded to multiples of a basic decamer (4.4 MDa) were detected. The appearance of unstained specimens of the cylindrical particles differs from negatively stained specimens. Viewed end-on the cylinders show no internal structure, but in well-preserved specimens cavities are apparent in the side views of the cylinders that resemble those seen in negatively stained specimens. Although they lack the characteristic "tiered" appearance, the number of decameric units can be counted and their arrangement within the particle seen.

Animals↗

PubMed1989

Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography.

During transcription in E. coli, the DNA-dependent RNA polymerase locates specific promoter sequences in the DNA template, melts a small region containing the transcription start site, initiates RNA synthesis, processively elongates the transcript, and finally terminates and releases the RNA product. Each step is regulated by interactions between the polymerase, the DNA, the nascent RNA, and a variety of regulatory proteins and ligands. The E. coli enzyme contains a catalytic core of two alpha-subunits, one beta- and one beta'-subunit, with relative molecular masses (Mr) of 36,512, 150,619 and 155,162, respectively. The holoenzyme has an additional regulatory subunit, normally sigma, of Mr 70,236. Preparations may also contain the omega-subunit (Mr approximately 10,000), which can be removed without affecting any known properties of the enzyme. Because the amino-acid sequences of the beta- and beta'-subunits are homologous to those of the largest subunits of the yeast, Drosophila and murine RNA polymerases, it seems likely that essential features of the three-dimensional structure and catalytic mechanism of RNA polymerase are also conserved across species. Crystals of RNA polymerase suitable for X-ray analysis have not yet been obtained, but two-dimensional crystals of E. coli RNA polymerase holoenzyme can be grown on positively charged lipid layers. Electron microscopy of these crystals in negative stain shows the enzyme in projection as an irregularly shaped complex approximately 100 x 100 x 160 A in size. We have now determined the three-dimensional structure by electron microscopy of negatively stained, two-dimensional crystals tilted at various angles to the incident electron beam. We find a structure in RNA polymerase similar to the active-site cleft of DNA polymerase I. In the light of functional similarities between these two enzymes, together with other evidence, this probably identifies the active-site region of RNA polymerase.

Bacterial Proteins↗

PubMed2003

Microsatellite instability and mutations in DNA mismatch repair genes in sporadic colorectal cancers.

PURPOSE: This study was designed to investigate the frequency of mutations in DNA mismatch repair genes in sporadic colorectal cancers. METHODS: Genomic DNAs procured from paraffin blocks of the pathologic specimens from 230 consecutive patients with colorectal cancer were examined for their microsatellite instability status using a mononucleotide microsatellite marker, BAT-26, and also evaluated expressions of hMLH1, hMSH2, and hMSH6 proteins by immunohistochemical staining. Any of these 230 patients did not have family histories of hereditary nonpolyposis colorectal cancer, familial adenomatous polyposis, colorectal cancer, or hereditary nonpolyposis colorectal-related cancers, such as endometrial, small bowel, and ureteral and renal pelvic cancers. When microsatellite instability was positive, mutations in the simple repeated sequences of TGF-betaRII, BAX, IGF IIR, hMSH3, and hMSH6 genes were examined. In microsatellite instability-positive or staining-negative cases, polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing detected mutations of hMLH1, hMSH2, and hMSH6 genes. If mutations were found in tumor tissue samples, we tested for a germline mutation with a microdissected corresponding normal tissue. RESULTS: Among 230 cases of sporadic colorectal cancer, 21 (9.1 percent) manifested microsatellite instability. In the immunohistochemical staining, 20 (8.6 percent) showed loss of expressions. All 20 staining-negative cases were microsatellite instability-positive. Only 1 of 21 (4.8 percent) microsatellite instability-positive cases showed intact staining for three proteins. The frame-shift mutations of the simple repetitive sequences were found in 17 cases (81.0 percent) in TGF-betaRII, 11 (52.4 percent) in BAX, 5 (23.8 percent) in IGF IIR, 7 (33.3 percent) in hMSH3, and 8 (38.1 percent) in hMSH6 genes. Germline mutation was observed in only one case, which accounts for 4.8 percent among positive microsatellite instability and 0.4 percent of total patients, and was found in hMSH2. Five somatic mutations (2 in hMLH1, 2 in hMSH2, and 1 in hMSH6) also were found. CONCLUSION: The results indicated that a germline mutation of DNA mismatch repair gene was a rare event in sporadic colorectal cancers.

Base Pair Mismatch↗

PubMed2004

Electron holography of non-stained bacterial surface layer proteins.

We report transmission electron microscopy (TEM) investigations on bacterial surface layers (S-layers) which belong to the simplest biomembranes existing in nature. S-layers are regular 2D protein crystals composed of single protein or glycoprotein species. In their native form, S-layers are weak phase objects giving only poor contrast in conventional TEM. Therefore, they are usually examined negatively stained. However, staining with heavy metal compounds may cause the formation of structural artefacts. In this work, electron microscopy studies of non-stained S-layers of Bacillus sphaericus NCTC 9602 were performed. Compared to other proteins, these S-layers are found relatively stable against radiation damage. Electron holography was applied where information about phase and amplitude of the diffracted electron wave is simultaneously obtained. In spite of small phase shifts observed, the phase image reconstructed from the hologram of the non-stained S-layer is found to be sensitive to rather slight structure and thickness variations. The lateral resolution, obtained so far, is less than that of conventional electron microscopy of negatively stained S-layers. It corresponds to the main lattice planes of 12.4 nm observed in the reconstructed electron phase image. In addition, as a unique feature of electron holography the phase image provides thickness information. Thus, the existence of double layers of the protein crystals could be easily visualized by the height profile of the specimen.

Bacillus↗

PubMed1994

Variable pitch in frozen-hydrated sickle hemoglobin fibers: an image analysis model study.

The intracellular polymerization of deoxyhemoglobin S (HbS) into helical fibers is the primary pathological event which gives rise to sickle cell disease. The structure of these fibers has previously been studied by electron microscopy of negatively stained specimens. We are extending these studies with unstained frozen-hydrated HbS fibers (cryo-EM), which afford better visualization of the internal details of the fiber structure than can be achieved by negative staining, but have lower signal-to-noise ratio images. The pitch of the HbS fiber structure varies locally along any given particle. Because rotation about the particle axis thus is partially decoupled from translation along the axis, the pitch and angular rotation of a fiber unit cell cannot be inferred by symmetry (as is the case with constant pitch helices). Image analysis procedures are presented which are capable of explicitly identifying the pitch and angular rotation of individual HbS fiber unit cells having low signal-to-noise ratios. Fiber images are divided into segments one unit cell long (63 A) which are analyzed in two steps. First each unit cell is aligned with constant pitch electron density reference models by cross-correlation. Correlation coefficients are then used to determine angular rotation and pitch. This procedure was tested, and found to be robust, using model images corrupted to simulate experimental problems normally encountered in the analysis of cryo-electron micrographs. The effects of limited resolution, low signal-to-noise ratio, scaling errors, and rotational and axial misalignment are described.

Computer Simulation↗

PubMed2001

The expression of cytoskeletal proteins (alpha-SMA, vimentin, desmin) in kidney tissue: a comparison of fetal, normal kidneys, and glomerulonephritis.

BACKGROUND: The aim of the study is a comparison of the expression of cytoskeletal proteins, alpha smooth muscle actin (alpha-SMA), vimentin, and desmin in fetal, normal kidney and proliferative (diffuse proliferative and membranoproliferative glomerulonephritis) and nonproliferative (membranous glomerulonephritis) glomerulonephritis. METHODS: We have studied the expression of cytoskeletal proteins (alpha-SMA, vimentin, desmin) in the paraffin embedded tissue sections from the kidneys of 10 normal kidney (adults and infants), 13 fetal kidney, 12 membranous glomerulonephritis (MGN), 8 membranoproliferative glomerulonephritis (MPGN), 8 diffuse proliferative glomerulonephritis (DPGN). Interstitial and glomerular positive stainings were evaluated. RESULTS: Vimentin expression was similar in normal infant and adult kidneys with positive staining in glomeruli and negative staining in interstitium. In fetal kidneys, glomerular mesangial and epithelial cells and blastematous areas showed positive reactivity with vimentin. Alpha-SMA staining was different among the groups. In fetal kidney, alpha-SMA expression was found in glomerular mesangial cells and blastematous areas. Alpha-SMA staining was positive in peritubular area and glomerular mesangial cells in infant kidney. In adult kidneys, glomerular staining with alpha-SMA disappeared but peritubular positivity continued. Interstitial staining with alpha-SMA was positive in fibrotic areas of proliferative (MPGN, DPGN) and non-proliferative (MGN) glomerulonephritis, but positive glomerular staining with alpha-SMA was found only proliferative glomerulonephritis. Desmin expression was negative in all groups. CONCLUSIONS: Desmin is not expressed in early stages of kidney growth, infant and adult kidneys, and proliferative and nonproliferative glomerulonephritis. Interstitial staining of vimentin in the diseased kidney tissues revealed increased fibrosis. Alpha-SMA revealed important differences in different stages of nephrogenesis. Glomerular mesangial staining with alpha-SMA in developing (fetal and infant kidneys) and proliferative glomerulonephritis suggest that it may be a marker of proliferation. In addition, it shows myofibroblastic differentiation in interstitium in diseased kidneys.

Actins↗