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PubMed1977

[Electron microscopic demonstration of viruses in the nasal secretion from infants].

Authors examined the nasal excretion of newborn and infant for the demonstration of viruses using negative-stain-electron-microscopic technique. The nasal cavity was rinsed with physiologic saline solution and the latter was examined. The negative stain technique is a relatively quick methode for the demonstration of viruses to support the suspition of the viral aetiology of a disease until serological diagnosis is available. Authors demonstrated Orthomyxoviruses, Adenovieuses, and Herpes-viruses in the nasal cavity.

Female↗
PubMed1997

[Detection of Mycobacterium tuberculosis in sputum using PCR and chemoluminescent display].

BACKGROUND: Polymerase chain reaction (PCR) using a probe with revealed chemoluminescence applied to the rapid diagnosis of pulmonary tuberculosis was used to detect Mycobacterium tuberculosis in sputum samples. METHODS: The sputum samples were treated with proteinase K, SDS, NaOH and CTAB. A fragment of the IS6110 sequence of Mycobacterium tuberculosis detected by a probe marked with digoxigenin and revealed chemoluminescence was amplified. RESULTS: The detection limit of the technique was situated at 10 mycobacteria per sample. Of 120 samples with a positive culture, 88 were positive by chemoluminescence probe (73.3%). The sensitivity decreased to 68.6% (35/51) in samples with negative staining and positive culture. CONCLUSIONS: The application of molecular biology techniques are a good diagnostic alternative in samples with negative staining and clinical suspicion of tuberculosis. Our system is a cheaper alternative than the commercial systems and is applicable in laboratories unable to use radioactive isotopes.

Bacteriological Techniques↗
PubMed1976

Negative silver staining in A-T and satellite DNA-rich regions of human chromosomes.

Human metaphase chromosomes were stained with silver following a pretreatment with a heated alkaline solution. The most conspicuous feature of the stained metaphases was the omission of silver staining in the secondary constrictions of chromosomes 1,9 and 16, and on the distal Yq. Our evidence indicates that the negative silver binding is due to the preferential removal or alteration of non-histone proteins associated with these regions. The cytochemical significance of these findings is discussed.

Adenine Nucleotides↗
PubMed1985

Sewage effluent biomonitoring. I. Survival, growth, and histopathological effects in channel catfish.

A 17-day, in situ, biomonitoring study using caged, juvenile channel catfish (Ictalurus punctatus) was conducted at five sites along a 9-km section of the Flint River at the Anthony Ragnone Wastewater Treatment Plant near Montrose, Michigan. Effects on survival, growth, and gill and liver histopathology were examined. No differences in growth, measured as wet weight gain, were observed between upstream control (UP) and experimental fish located 9 km downstream from the outfall (Down 4). Acute, 100% mortality occurred at study sites 300 and 500 m downstream from the outfall (Down 1 and Down 2, respectively). Mortality was probably due to excessive mean total residual chlorine concentration (0.24 and 0.30 mg/liter at Down 1 and Down 2, respectively). No mortality was observed at the remaining study sites. Forty percent of the fish at UP had slight hyperplasia and hypertrophy of the respiratory epithelium. All fish at Down 4 had two or more moderate to severe histopathological damages to the gills, including severe hyperplasia of the epithelial cells, clubbing and fusion of the secondary lamellae, moderate to severe edema in the secondary lamellae, and multiple, blood-filled aneurysms. Thirty-eight percent of livers from the UP fish were extremely vacuolated compared to 60% of the Down 4 fish livers. Both the vacuolated and less vacuolated (dense) liver sections stained negatively for lipids with oil red O. The vacuolated liver sections were qualitatively more intense than the dense sections when stained with Best's carmine. Vacuolated liver sections stained negatively with Best's carmine when pretreated with amylase, thereby indicating that the vacuolation represented glycogen storage. There was no correlation between the histopathological changes and any deleterious effects on growth or survival during the 17-day exposure. However, the gill histopathological changes would likely increase susceptibility to bacterial gill disease, and cause a reduction in the scope for activity due to impaired oxygen diffusion in the gills.

Animals↗
PubMed1975

Appearances of microtubules after various fixative procedures, and comparison with the appearances of tobacco mosaic virus.

Microtubules in crane-fly spermatids appeared altered when the glutaraldehyde-fixed cells were not postfixed with osmium tetroxide. The cytoplasmic microtubules were altered more than the doublet microtubules. Addition of osmium tetroxide after dehydration did not produce appearances identical with those of microtubules postfixed directly after glutaraldehyde, and thus at least some alterations occurred during dehydration, possibly due to extraction of microtubule-associated lipid. The omission of osmium tetroxide postfixation did not cause drastic alterations in the appearances of either tobacco mosaic virus (TMV), or polymerized tobacco mosaic virus protein (without RNA), suggesting that microtubule stability is different from TMV stability (with respect to the embedment procedure). The electron-dense stain associated with embedded-sectioned TMV is predominantly outside the TMV protein, as demonstrated by the known distribution of TMV protein compared with the dimensions of sectioned TMV and negatively stained TMV. The same might hold true for microtubules, as evidenced by the dimensions of negatively stained, isolated brain microtubules compared with those of embedded and sectioned brain microtubules.

Acetates↗
PubMed1980

Methods for scanning and transmission electron microscopy of normal and damaged gram-negative bacteria.

Preparatory procedures and different types of electron microscopical imaging of bacterial specimens are described. Transmission electron microscopy (TEM) was performed on ultra-thin sections of plastic embedded material and negatively stained specimens. Metal coated, whole bacteria were prepared for scanning electron microscopy (SEM). The rapid procedure for TEM observation of negatively stained specimens indicates size, shape and appendages. TEM micrographs of ultra-thin sections show details of the cell wall and internal structures of the cytoplasm. SEM is superior in demonstrating size and gross surface morphology. The three methods are complementary in the study of bacterial cell ultrastructural morphology.

Bacteria↗
PubMed1988

Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7.

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.

Amino Acid Sequence↗
PubMed1982

Mass mapping of a protein complex with the scanning transmission electron microscope.

A mass map of the hexagonally packed intermediate layer (HPI-layer), a regular protein monolayer from the cell envelope of Micrococcus radiodurans, has been obtained by scanning transmission electron microscopy. Samples were freeze-dried within the microscope, and low-dose images were recorded in the dark-field mode directly in digital form and processed by correlation averaging. The averaged projection of the unstained structure--i.e., the mass map--thus calculated shows a resolution to 3-nm period and reveals morphological features consistent with those obtained by negative staining. The mass of individual morphological domains was extracted by using variously the mass map itself or an average from a negatively stained HPI layer to define the domain boundaries. Protrusions as small as 1,300 daltons could be measured reproducibly within the unit cell of 655,000 daltons. The method developed opens an avenue to identify molecular species in situ and to correlate topographic information with biochemical data.

Bacterial Proteins↗
PubMed1981

Phycobilisome-thylakoid Topography on Photosynthetically Active Vesicles of Porphyridium cruentum.

Conditions are described for isolating functional phycobilisome-thylakoid vesicles from the red alga Porphyridium cruentum. Phycobilisome-thylakoid vesicles were prepared by brief sonication and centrifugation in a medium containing 0.5 molar sucrose, 0.5 molar potassium phosphate, and 0.3 molar sodium citrate (pH 7.0). They required ferricyanide as an oxidant and had O(2) evolution rates (about 450 micromoles O(2) per hour per milligram chlorophyll) higher than whole cells (about 250 micromoles O(2) per hour per milligram chlorophyll). Energy transfer to photosystem II chlorophyll was evident from a high F695 nanometer (-196 C) emission peak. Preparations could be stored for over 24 hours and were considerably more stable than those from the cyanobacterium Anabaena variabilis (Katoh T, E Gantt 1979 Biochim Biophys Acta 546: 383-393). In electron micrographs of negatively stained material, the active thylakoid vesicles were found covered by closely spaced phycobilisomes on their external surface. The phycobilisome number in negatively stained vesicles was 450 per square micrometer, which was in the same range as the 400 per square micrometer observed in surface sections. A cell containing 1.5 x 10(-6) micrograms phycoerythrin and 1.3 x 10(-6) micrograms chlorophyll was found to contain 5 to 7 x 10(5) phycobilisomes on a thylakoid area of 1.1 to 1.6 x 10(3) square micrometers.

Journal Article↗
PubMed2002

Expression of p53 gene product and cell proliferation marker Ki-67 in Ewing's sarcoma: correlation with clinical outcome.

Overexpression of tumor suppressor gene p53, cell proliferation nuclear antigen Ki-67, and proto-oncogene HER-2/neu are associated with poor prognosis in some tumors. We studied p53, Ki-67, and HER-2/neu immunohistochemical expression in archival biopsies of 37 patients with Ewing's sarcoma (ES). Patients with ES were treated at four Israeli hospitals between 1982 and 2000. Formalin-fixed paraffin-embedded tissue sections were stained by immunohistochemistry for p53, Ki-67, and HER-2/neu. More than 300 cells were counted on each slide, and the percentage of positively stained nuclei was computed. p53 overexpression was defined as nuclear staining of >2.3% of cells, Ki-67 overexpression as nuclear staining of >8.3% malignant cells. HER-2/neu staining was scored semiquantitatively on a scale of 0 to 4+. Twenty-two of 37 patients are alive and well, with mean follow-up time of 38 months. There was overexpression of p53 in 16 patients (43%) and of Ki-67 in 21 patients (57%). The correlation between p53 and Ki-67 overexpressions was 0.61. We found no overexpression of HER-2/neu. Median relapse-free survival (RFS) was statistically significantly shorter for patients with p53 overexpression (25 months) than for patients with negative staining (>92 months). The prognostic value of p53 overexpression was also significant after adjusting for tumor location and age. Median RFS was shorter for patients with positive Ki-67 staining (40 months) than for patients with negative staining (80 months) but did not reach statistical significance. Our study suggests that p53 is a predictor of RFS in patients with ES. More patients must be studied to assess the validity of this observation.

Adolescent↗
PubMed2006

Isolation of an ethanol-tolerant endospore-forming Gram-negative Brevibacillus sp. as a covert contaminant in grape tissue cultures.

AIMS: To characterize the alcohol-surviving bacterial isolate ARBG1 from in vitro grapes (Vitis vinifera). METHODS AND RESULTS: Two bacterial strains that survived in covert form in grape cultures were isolated from the spent alcohol used for disinfecting the tools of which one (ARBG2) was characterized earlier. The present study describes characterization of the second isolate, ARBG1. Nutrient agar (NA)-derived colonies of ARBG1 displayed consistently Gram-negative staining rods (2-4x0.5-0.6 micro) substantiated by KOH mucoid thread test. Older cultures (3-7 days) showed emergence of Gram-negative staining, oblong, phase-refractile cells with ellipsoidal spores. The growth and sporulation were modified by growth medium and incubation temperature with the optimum around 37 degrees C. Identification attempts involving microscopic, biochemical, Biolog or fatty acid profiling approaches brought in mixed and inconclusive results. PCR amplification of 16S rDNA was not successful with the standard primers 27F and 1492R but with 27F and a modified primer ARBG1-RP1. The identity of the isolate was established as Brevibacillus sp. based on partial 16S rDNA sequence data from eight single colonies with Gram-positive Brevibacillus choshinensis as the closest match (99.5%). Spotting tests on NA employing spore suspension in aqueous ethanol (0%, 25%, 50%, 60%, 70%, 80% or 90%, v/v) indicated unhindered bacterial-survival in alcohol for 1 month, and that at 2 or 4 months revealed 90% ethanol as more sporicidal than lower levels, corroborated by plating results. Grape microcuttings inoculated with ARBG1 showed substantial general colonization of shoots, roots and medium but low endophytic colonization. CONCLUSIONS: The rare type of spore-producing consistently Gram-negative bacterial isolate ARBG1 was identified as Brevibacillus sp. based on 16S rDNA sequence similarity. The alcohol-defying organism was nonpathogenic and survived in covert form in grape cultures. Aqueous 90% ethanol appeared more sporicidal than lower levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Characterization of an unusual endospore-forming Gram-negative bacterium, observation that some bacteria may fall outside the purview of standard 16S rDNA primers, elucidation of the threats of covert bacteria in plant tissue cultures and alcohol-mediated lateral transmission of spore formers, and the revelation that 70-80% ethanol may not be the most effective bactericidal concentration for all bacteria.

Base Sequence↗
PubMed1984

Effects of fixing and staining on images of tropomyosin Mg-paracrystals.

Tropomyosin Mg-paracrystals have been studied by positive and negative staining with uranyl acetate and sodium phosphotungstate, either unfixed or fixed with glutaraldehyde. Fixation causes changes in some of the paracrystal bands. Some of the bands are more intense when a positively-charged staining ion is used than when a negatively-charged ion is used, in both negative and positive-staining techniques. This result rules out the suggestion that negative staining is not affected by the charge of the stain in tropomyosin, and agrees with findings in other specimens. Therefore it is unlikely that any simple parameter based on residue size can be used to predict the image intensity from the amino acid sequence for tropomyosin. These staining patterns cast doubt on the earlier interpretation of a prominent white band in the paracrystal as an overlap of alpha-helical molecular ends.

Animals↗
PubMed1985

Crystallization of the Ca2+-ATPase of sarcoplasmic reticulum by calcium and lanthanide ions.

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop in sarcoplasmic reticulum vesicles exposed to Ca2+ or lanthanide ions. The Ca2+- or lanthanide-induced crystals are presumed to represent the E1 conformation of the Ca2+-ATPase, and their crystal form is clearly different from the earlier described E2 crystals induced by Na3VO4 in the presence of ethylene glycol bis(beta aminoethyl ether)-N,N,N',N'-tetraacetic acid (Taylor, K. A., Dux, L., and Martonosi, A. (1984) J. Mol. Biol. 174, 193-204). Analysis of the crystalline arrays by negative staining or freeze-fracture electron microscopy reveals obliquely oriented rows of particles corresponding to individual Ca2+-ATPase molecules. Computer analysis of the negatively stained lanthanide-induced crystalline Ca2+-ATPase arrays shows that the molecules are arranged in a P1 lattice. The pear-shaped profiles of Ca2+-ATPase molecules seen in projection in the density maps are similar to those seen in vanadate-induced crystals. The space group and unit cell dimensions of the E1 crystals are consistent with Ca2+-ATPase monomers as structural units, while the vanadate-induced E2 crystals form by lateral aggregation of chains of Ca2+-ATPase dimers. The transition between the E1 and E2 conformations may involve a shift in the monomer-oligomer equilibrium of the Ca2+-ATPase. The formation of E1 crystals by PrCl3 is promoted by inside negative membrane potential, presumably through stabilization of the E1 conformation of the enzyme. Cleavage of the Ca2+-ATPase by trypsin into two major fragments (A and B) did not interfere with the Ca2+- or the Pr3+-induced crystallization.

Adenosine Triphosphate↗
PubMed2003

Three-dimensional reconstruction of hibiscus chlorotic ringspot virus.

Hibiscus chlorotic ringspot virus (HCRSV) is a positive-sense, single-stranded RNA virus, which belongs to the Tombusviridae family and infects plants of the Hibiscus genus, including kenaf, a woody plant of agricultural importance. These icosahedral viruses have a capsid consisting of 180 copies of coat protein (CP) arranged with T=3 symmetry. The CP consists of an internal RNA-binding domain, a shell-forming domain and a protruding domain. The HCRSV virion was reconstructed to about 12A resolution from cryo-EM images using the program EMAN. The structure had the arrangement of 90 dimers of protruding domains characteristic of the Tombusviridae. Reconstructions were also made from negatively stained samples, and showed essentially the same features. In addition, a particle of a different, "smooth" appearance was also identified in the negatively stained samples. These particles were slightly smaller and lacked protruding domains. Biochemical analysis confirmed the presence of two protein products: a 37 kDa protein identified as HCRSV CP and a 54 kDa protein that appeared to be of non-HCRSV origin.

Amino Acid Sequence↗
PubMed1990

Bundling of actin filaments by alpha-actinin depends on its molecular length.

Cross-linking of actin filaments (F-actin) into bundles and networks was investigated with three different isoforms of the dumbbell-shaped alpha-actinin homodimer under identical reaction conditions. These were isolated from chicken gizzard smooth muscle, Acanthamoeba, and Dictyostelium, respectively. Examination in the electron microscope revealed that each isoform was able to cross-link F-actin into networks. In addition, F-actin bundles were obtained with chicken gizzard and Acanthamoeba alpha-actinin, but not Dictyostelium alpha-actinin under conditions where actin by itself polymerized into disperse filaments. This F-actin bundle formation critically depended on the proper molar ratio of alpha-actinin to actin, and hence F-actin bundles immediately disappeared when free alpha-actinin was withdrawn from the surrounding medium. The apparent dissociation constants (Kds) at half-saturation of the actin binding sites were 0.4 microM at 22 degrees C and 1.2 microM at 37 degrees C for chicken gizzard, and 2.7 microM at 22 degrees C for both Acanthamoeba and Dictyostelium alpha-actinin. Chicken gizzard and Dictyostelium alpha-actinin predominantly cross-linked actin filaments in an antiparallel fashion, whereas Acanthamoeba alpha-actinin cross-linked actin filaments preferentially in a parallel fashion. The average molecular length of free alpha-actinin was 37 nm for glycerol-sprayed/rotary metal-shadowed and 35 nm for negatively stained chicken gizzard; 46 and 44 nm, respectively, for Acanthamoeba; and 34 and 31 nm, respectively, for Dictyostelium alpha-actinin. In negatively stained preparations we also evaluated the average molecular length of alpha-actinin when bound to actin filaments: 36 nm for chicken gizzard and 35 nm for Acanthamoeba alpha-actinin, a molecular length roughly coinciding with the crossover repeat of the two-stranded F-actin helix (i.e., 36 nm), but only 28 nm for Dictyostelium alpha-actinin. Furthermore, the minimal spacing between cross-linking alpha-actinin molecules along actin filaments was close to 36 nm for both smooth muscle and Acanthamoeba alpha-actinin, but only 31 nm for Dictyostelium alpha-actinin. This observation suggests that the molecular length of the alpha-actinin homodimer may determine its spacing along the actin filament, and hence F-actin bundle formation may require "tight" (i.e., one molecule after the other) and "untwisted" (i.e., the long axis of the molecule being parallel to the actin filament axis) packing of alpha-actinin molecules along the actin filaments.

Acanthamoeba↗
PubMed1989

An ultrastructural study of crossbridge arrangement in the fish skeletal muscle thick filament.

A procedure has been developed for isolating gold-fish skeletal muscle thick filaments that preserves the near-helical arrangement of the myosin cross-bridges under relaxing conditions. These filaments have been examined by electron microscopy and computer image analysis. Electron micrographs of the negatively stained filaments showed a clear periodicity associated with the crossbridges, with an axial repeat every 42.9 nm. Computed Fourier transforms of the negatively stained filaments showed a series of layer lines confirming this periodicity, and were similar to the X-ray diffraction patterns of fish muscle obtained by J. Hartford and J. Squire. Analysis of the computed transform data and filtered images of the isolated fish filaments demonstrated that the myosin crossbridges lie along three strands. Platinum shadowing demonstrated that the strands have a right-handed orientation, and computed transforms and filtered images of the shadowed filaments suggest that the crossbridges are perturbed both axially and azimuthally from an ideal helical arrangement.

Animals↗
PubMed1991

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense.

Thin sections, freeze-etched, and negatively stained preparations of Methanocorpusculum sinense cells reveal a highly lobed cell structure with a hexagonally arranged surface layer (S layer). Digital image processing of negatively stained envelope fragments show that the S layer forms a porous but strongly interconnected network. Since the S layer is the exclusive cell envelope component outside the cytoplasmic membrane it must have a cell shape determining and maintaining function. Although lattice faults such as disclinations and dislocations are a geometrical necessity on the surface of a closed protein crystal, our data indicate that they also play important roles as sites for the incorporation of new morphological units, in the formation of the lobed cell structure, and in the cell division process. In freeze-etched preparations of intact cells numerous positive and negative 60 degree wedge disclinations can be detected which form pentagons and heptagons in the hexagonal array. Complementary pairs of pentagons and heptagons are the termination points of edge dislocations. They can be expected to function both as sites for incorporation of new morphological units into the lattice and as initiation points for the cell division process. The latter is determined by the ratio between the increase of protoplast volume and the increase in actual S-layer surface area during cell growth. We postulate that this mode of cell fission represents a common feature in lobed archaebacteria which possess an S layer as the exclusive wall component.

Archaea↗
PubMed2004

Expression of p63 (TA and deltaN isoforms) in human primary well differentiated buccal carcinomas.

Abnormalities in the p53 gene have been regarded as the most consistent genetic abnormalities detected in head and neck squamous cell carcinogenesis. Two new members of the p53 gene family, p73 and p63, have recently been identified. We investigated the expression of the two N-terminal p63 isoforms (TA and deltaN isoforms) in human primary well-differentiated buccal squamous cell carcinoma. Both TAp63 and deltaNp63 isoforms were detected in the basal/suprabasal layers of all of the five specimens of normal buccal mucosa. The deltaNp63 isoform was found in all of the 23 specimens of human primary well-differentiated buccal carcinoma whereas TAp63 isoform was absent in 18 (78.3%) of the 23 specimens. The immunostaining patterns of both TAp63 and deltaNp63 isoforms were similar in that the p63 positivity was noted mainly in the peripheral cells of tumor nests whereas negative staining was observed in the areas with keratin pearl formation. A higher number of T3-T4 patients and patients with recurrence showed negative staining of TAp63 than T1-T2 patients and patients without recurrence but the difference was not statistically significant. These results suggested that specific p63 isoforms were associated with human oral squamous cell carcinogenesis. The deltaNp63 isoforms might be involved in epithelial differentiation and proliferation in human oral carcinogenesis whereas there was evidence for a possible role of TAp63 under-expression in human oral tumorigenesis.

Adult↗