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PubMed1985

Correlation between degradation and ultrastructure of peptidoglycan during autolysis of Escherichia coli.

The kinetics of peptidoglycan degradation were examined under different conditions of autolysis of Escherichia coli. With cephaloridine- or moenomycin-induced autolysis, degradation did not exceed 25 to 35%, whereas in EDTA-induced autolysis it rapidly reached 65 to 70%. When nonautolyzing cells were fixed overnight with glutaraldehyde, followed by an osmium fixation, and thin sections were stained by the phosphotungstic acid method, a dark, 15-nm-thick layer of uniform appearance and constant width occupied the whole area between the inner and outer membranes of the envelope. The stained material was tentatively identified with peptidoglycan. Ultrastructural changes in this phosphotungstic acid-stained periplasmic space were investigated at different time intervals after induction of autolysis. In all cases, breakdown proceeded over the whole cell surface. During antibiotic-induced autolysis a progressive thinning down limited to the inner side of the layer was observed. During EDTA-induced autolysis, the rapid decrease in thickness correlated well with the important loss of material labeled with [3H]diaminopimelic acid. Considering these changes and the insufficient amounts of peptidoglycan (1.3 U/nm2) necessary to account for a regularly structured polymer occupying the whole 15-nm layer, it was speculated that peptidoglycan might be unevenly distributed throughout the periplasmic space.

Anti-Bacterial Agents↗
PubMed1987

Presence of basic proteins and ribonucleoproteins in the neck region of human spermatids and spermatozoa.

The ultrastructural cytochemical study of the neck region of human spermatids and spermatozoa revealed that, besides the centrioles, the basal plate, the lateral cupshaped junction body and the annulus material showed affinity for both ethylene diamine tetra-acetic acid (EDTA) stain (a preferential staining method for ribonucleoproteins) and ethanolic phosphotungstic acid (PTA) stain (for cellular localisation of basic proteins). The capitulum and the surface of the striated columns were also stained with ethanolic PTA. The chromatoid body and annulate lamellae-which were frequently encountered in the neck region--stained with both EDTA and ethanolic PTA techniques. This suggests a possible participation of the chromatoid body and annulate lamellae in the formation of the neck region structures.

Adult↗
PubMed1986

Alterations in the sialic acid content of the rat glomerular filter in aminonucleoside nephrosis.

A daily injection protocol with puromycin aminonucleoside (PAN) causes loss of sialic acid from the glomerular filter. These changes have been studied previously by colloidal iron staining, but we have recently shown that phosphotungstic acid (PTA) at low pH allows the demonstration of sialic acid groups in the glomerular basement membrane in ultrathin sections of glycolmethacrylate(GMA)-embedded rat kidney. With this technique the slit diaphragm is seen as a continuation of the luminal cell coat and the method also gives an idea of the sialic acid distribution at the podocyte plasma membrane. The availability of this method made it possible to reevaluate the results obtained earlier in aminonucleoside (PAN) nephrosis indicating a decrease in the sialic acid content of the glomerulus. Although there are changes in the epithelial architecture, the ultrastructural appearances of the basement membrane are only slightly altered in PAN nephrosis. Detachment of epithelial cells was variable in different animals. Seven days after the first injection of PAN, staining with PTA revealed local defects in the lamina rara externa which later became more extensive. In PAN-treated animals the luminal cell coat showed reduced staining and large areas of the plasma membrane were completely devoid of a cell coat. These changes coincided with the onset of heavy proteinuria. The results indicate that both the basement membrane and the epithelial plasma membrane are affected in PAN nephrosis, as revealed by decreased staining for sialic acid-containing molecules in the basement membrane and by changes in the epithelial cell coat. The defects in the cell coat material point to functional alterations at the level of the slit pores and it is suggested that the decrease in sialic acid content of the lamina rara externa may be partly responsible for defects in the size-selective filtration barrier in PAN nephrosis.

Animals↗
PubMed1986

Ultrastructural and cytochemical study of the pinealocyte nucleolus in rats.

The pinealocyte nucleolus has practically not been studied, although many studies have been carried out on this particular cell. In order to study the pinealocyte nucleolus, we have used conventional electron microscopy techniques, as well as cytochemical methods: silver nitrate, phosphotungstic acid (PTA) and ethylenediaminetetraacetic acid (EDTA). The pinealocyte nucleolus shows a reticular structure formed by a dense fibrillar component and closely packed granules (granular component), as well as electron lucent spaces which contain nucleoplasm. There are also places of light fibrillar material (fibrillar centers). Using the Ag-staining we have observed spherical aggregates of argyrophylic granules densely packed (fibrillar centers) and other granules loosely packed in its periphery (dense fibrillar component). PTA stains preferentially the fibrillar centers. We have identified as heterochromatin a dense granular aggregate that appears associated to the nucleolus. We present the pinealocyte nucleolus as an excellent model for the study of the functional meaning of nucleolar components.

Animals↗
PubMed1987

Ultrastructural alterations in the sialic acid distribution in minimal change disease and membranous glomerulonephritis.

Kidney biopsy specimens from patients with minimal change disease and membranous glomerulonephritis were embedded in glycolmethacrylate and stained with phosphotungstic acid (PTA) at low pH. Biopsy specimens from patients without proteinuria served as a control. The PTA staining at low pH on glycolmethacrylate sections was used to study the changes in the sialic acid content of the lamina rara externa of the glomerular basement membrane. This method also gives a clear picture of the changes occurring at the epithelial cell coat and these alterations have implications on the distribution of the negative charges. In minimal change disease no alterations could be observed in the sialic acid content of the lamina rara externa. But the luminal epithelial cell coat showed obvious changes in conjunction with extensive foot process widening. In membranous glomerulonephritis with heavy deposits the staining of the lamina rara externa became almost completely negative and the foot process architecture was strongly affected. Obvious defects at the luminal epithelial cell coat, as observed in minimal change disease, were also found regularly. The alterations at the epithelial cell coat are tentatively related to the selective proteinuria reported in minimal change disease. In addition the non-selective proteinuria observed in non-minimal glomerulopathies, may find its origin in the absence of sialic acid molecules from the lamina rara externa.

Basement Membrane↗
PubMed1989

Ethidium bromide- and propidium iodide-PTA staining of nucleic acids at the electron microscopic level.

Ultra-thin sections of various tissues were stained with ethidium bromide or propidium iodide, two fluorescent markers widely used for quantitation of nucleic acids. The fluorochromes, tested at different concentrations, were then revealed by incubation of the sections with neutralized phosphotungstic acid. We showed that at the electron microscopic level only nucleic acid-containing structures are revealed. Chromatin, nucleolus, and ribosomes appear to be stained by the end-product of the reaction. Furthermore, controls with proteases and nucleases showed that the staining is related to the binding of the fluorochromes to DNA and RNA and to the subsequent detection of the dyes by neutralized PTA.

Animals↗
PubMed1980

A search for protein cores in chromosomes: is the scaffold an artifact?

A protein chromosome scaffold structure has been proposed that acts as a structural framework for attachment of chromosomal DNA. There are several troubling aspects of this concept: (1) such structures have not been seen in many previous thin-section and whole-mount electron microscopy studies of metaphase chromosomes, while they are readily seen in leptotene and zygotene chromosomes; (2) such a structure poses problems for sister chromatid exchanges; and (3) the published photographs show a marked variation in the amount of scaffold in different whole-mount preparations. An alternative explanation is that the scaffold in whole-mount preparations represents incomplete dispersion of the high concentration of chromatin in the center of chromosomes, and when the histones are removed and the DNA dispersed, the remaining nonhistone proteins (NHPs) aggregate to form a chromosome-shaped structure. Two studies were done to determine if the scaffold is real or an artifact: (1) Chinese hamster mitotic cells and isolated chromosomes were examined using two protein stains -EDTA-regressive staining and phosphotungstic acid (PTA) stain. The EDTA-regressive stain showed ribonucleoprotein particles at the periphery of the chromosomes but nothing at the center of the chromosomes. The PTA stain showed the kinetochore plates but no central structures; and (2) isolated chromosomes were partially dispersed to decrease the high concentration of chromatin in the center of the chromosome, then treated with 4 M ammonium acetate or 2 M NaCl to dehistonize them and disperse the DNA. Under these circumstances, no chromosome scaffold was seen. We conclude that the scaffold structure is an artifact resulting from incomplete dispersion of central chromatin and aggregation of NHPs in dehistonized chromosomes.

Animals↗
PubMed1978

The packaging unit: a basic structural feature for the condensation of late cricket spermatid nuclei.

The alignment, folding and packaging of cricket chromatin was examined during late spermiogenesis by an electron-microscope study of nuclei dispersed by air--liquid surface tension forces after detergent treatment. Late developing spermatid genomes arrange themselves in multiple packaging units in a stepwise process which includes: (1) a loss of the beaded repeating structure of chromatin as nucleoprotein fibres become smooth and gradually assume a uniform diameter; (2) a side-by--side alignment of structurally modified chromatin fibres; and (3) a regular folding into packaging units. Alignment and folding of chromatin fibres are presumably mediated by intermolecular bonds easily disrupted by spreading forces. In very late spermatids, interfibre binding forces are difficult to overrride by spreading alone, indicating a stronger cross-linking of increasingly coalescent packaging units. 'Unit to unit' coalescence stabilizes the nuclear structure, first limiting and afterwards denying penetration of phosphotungstic acid, as displayed in thin sections of extremely cricket spermatid nuclei. Binding of phosphotungstate by nuclear basic proteins can be facilitated by limited protein solubilization after disulphide reduction of unfixed cricket tests with sodium dodecyl sulphate and dithiothreitol. Results of this study permit the proposal of model experiments useful for clarifying the organization of highly condensed spermatid genomes and for evaluating the structure of genome segments in systems wherein changes of chromatin-associated protein occur.

Animals↗
PubMed1989

Ultrastructural histochemistry of mesotheliomas and adenocarcinomas in malignant effusions.

The results of electron microscopic examination of cytologic specimens from six cases of mesothelioma and 10 cases of metastatic carcinoma of different origins are presented. The formation of cell clusters in malignant effusions from the two neoplasms has been thoroughly investigated: in mesotheliomas, cells had longer, more slender microvilli than in carcinomas and more abundant bundles of intermediate filaments; the central cavity often seen in the clusters frequently contained collagen and showed basement membrane production. The application of periodic acid-silver methenamine (PASM) and phosphotungstic acid (PTA) demonstrated a peculiar ultrastructural difference in cell coat staining in the two tumor types: in mesotheliomas, PTA and PASM were consistently negative along the outer surface of the cell aggregates, while carcinomas displayed a positive reaction either on the outer surface or on both inner and outer surfaces of the clusters. The diagnostic significance of the above-mentioned difference between the two neoplasms will require further investigation in a larger number of cases.

Adenocarcinoma↗
PubMed1995

Melanotic ependymoma in a Goeldi's marmoset (Callimico goeldii).

A spontaneous melanotic ependymoma was observed in the brain of an adult female Goeldi's marmoset (Callimico goeldii). The mass completely occupied the left lateral ventricle, rupturing the fornix and corpus callosum, and compressing the adjacent neuropil. Special histochemical techniques, including melanin bleach, periodic acid-Schiff, Perls iron and phosphotungstic acid hematoxylin, demonstrated the neoplasms to be an ependymoma with a rare melanotic differentiation.

Animals↗
PubMedDate not supplied

Immunohistochemical and ultrastructural observations on adenoid cystic carcinoma of salivary glands. With special reference to intermediate filaments and proteoglycan particles.

In adenoid cystic carcinoma (ACC) of salivary gland origin, intermediate filaments of tumour cells were investigated by immunohistochemical techniques and complex carbohydrates within the pseudocysts by ultrastructural methods. The intermediate filament proteins vimentin and cytokeratin were found in most neoplastic cells. The pseudocysts of the tumours were found to contain proteoglycan particles stainable after glutaraldehyde fixation--but not after osmium fixation alone. Ruthenium red (cationic dye) enhanced the staining whereas periodic acid-chromic acid-silver methenamine staining or phosphotungstic acid staining did not visualize the proteoglycan particles mainly containing acid proteoglycans which are normally produced by mesenchymal tissue. The cells of adenoid cystic carcinoma thus have an intermediate filament content and produce complex carbohydrates characteristic of both epithelial and mesenchymal tissue. Hence, an origin in an undifferentiated pluripotential cell seems plausible.

Carcinoma, Adenoid Cystic↗
PubMed1984

A new technique for studying the relationship between maternal diabetes and the sialic acid content of fetal pulmonary surfactant.

By adapting a standard method for precipitation of high-density lipoprotein cholesterol with phosphotungstic acid (PTA) and Mg2+, fetal pulmonary surfactant can be rapidly isolated from human amniotic fluid, 97% of the total disaturated phosphatidylcholine being precipitated from the sample. The lecithin/sphingomyelin ratio for 17 separate specimens correlated reasonably well (r = 0.76) with the concentration of disaturated phosphatidylcholine in the PTA precipitate. Using thiobarbituric acid as the chromophore, I measured sialic acid in the PTA precipitate after overnight treatment with neuraminidase. The sialic acid/protein ratio for the PTA precipitate was identical to that for the surfactant, as isolated by ultracentrifugation. The concentrations of insulin and C-peptide were significantly greater in specimens of amniotic fluid from mothers with diabetes than from non-diabetic mothers (p less than 0.001). When the specimens were segregated according to a C-peptide cutoff value of 4 micrograms/L, there was a small, significant decrease in PTA-precipitated concentrations of sialic acid in the samples with C-peptide greater than 4 micrograms/L. The results suggest a possible mechanism for the increased incidence of respiratory distress among infants born to diabetic mothers.

Amniotic Fluid↗
PubMed1999

A new liquid homogeneous assay for the determination of HDL-cholesterol. A comparison to precipitation with phosphotungstic acid/MgCl2 and a lyophilized homogeneous assay.

We evaluated a new ready to use liquid assay for the homogeneous determination of HDL-cholesterol (HDL-C; Merck, Darmstadt, Germany) in comparison to phosphotungstic acid precipitation and a homogeneous assay, based on sulfated alpha-cyclodextrin and polyethylene glycol-modified enzymes (Roche Diagnostics/Boehringer Mannheim, Germany). The new liquid homogeneous HDL-C assay had inter-assay coefficients' of variation of less than 2.1%. The method is linear up to at least 3.11 mmol/I HDL-C, but even at 4.40 mmol/I the deviation from the expected value is less than 5%. Spinking experiments with low density lipoproteins and very low density lipoproteins proved that the new assay was specific for high density lipoproteins up to cholesterol associated with low density lipoproteins (LDL-C) and very low density lipoproteins (VLDL)-triglyceride concentrations of 18.13 and 22.60 mmol/l, respectively. Free fatty acids above 2mmol/l did not interfere. Icteric samples with bilirubin concentrations between 170 and 400 micromol/l did not show any systematic deviation compared to the precipitation procedure. In addition, serum hemoglobin concentrations up to 7.0 mmol/l and ascorbic acid up to 3000 micromol/l did not interfere with the HDL-C assay. An intermethod comparison including 120 samples revealed good agreement of the liquid HDL-C assay and the precipitation procedure (y = 0.943x + 0.074 mmol/l; r = 0.992). The new homogeneous HDL-C assay is thus precise, comparable and robust. Due to its ease of handling this assay will significantly facilitate attempts to include the differentiation between HDL-C and LDL-C in the routine screening for cardiovascular risk factors and in the monitoring of lipid lowering therapy.

Chemical Precipitation↗
PubMed2001

Ultrastructural and immunocytochemical characterization of interstitial cells in pre- and postnatal developing sheep pineal gland.

Pineal gland interstitial cells from 32 sheep embryos (from day 54 of gestation until birth) and 18 sheep (from 1 month to >2 years) were analysed using ultrastructural and immunohistochemical techniques. From day 98 of gestation and throughout postnatal development, a second cell type was observed in addition to pinealocytes; these cells displayed uniform ultrastructural features similar to those of CNS astrocytes. Ultrastructural homogeneity was not matched by the results of histochemical and immunohistochemical analysis. Expression of phosphotungstic acid hematoxylin, glial fibrillary acidic protein and vimentin indicates that the second cell population in the developing ovine pineal gland is, in fact, a combination of glial-astrocyte cells at varying stages of maturity. Pineal interstitial cells started to show signs of functional activity evident in vascular tropism; such activity, evident from around day 98 of gestation, appeared to relate to the exchange of substances between the pineal parenchyma and blood vessels and, though it continued throughout postnatal development, was most evident in animals slaughtered between 9 months and 2 years of age (group II). Morphologically, functional activity in interstitial cells in this age-group was apparent in: 1, formation of specific contact sites between interstitial cells and nerve fibres in the perivascular space; and 2, the presence of numerous gap junctions between the bulbous endings of cytoplasmic processes.

Animals↗
PubMed1977

Electron histochemistry of mucosubstances in normal human rectal epithelium.

The electron microscopic histochemistry of mucosubstances in sigmoidoscopically and microscopically normal rectal biopsies was studied using techniques currently available. The deposition of Alcian Blue and Ruthenium Red and the distribution of Concanavalin A receptors were limited to the epithelial cell borders. Mucosubstances in the fuzzy coat, Golgi apparatus, lysosomes and secretory vesicles were demonstrated by the periodic acid-chromic acid oxidation methods. Glycogen was demonstrated in the epithelium by periodate oxidation methods and the complex cyanide technique. There was little difference in the distribution of mucosubstances in the epithelial cells at any level of the crypts. Phosphotungstic acid staining under controlled conditions gave a similar distribution of mucosubstances to those revealed by the oxidation techniques.

Alcian Blue↗
PubMed1964

INTERNAL STRUCTURES OF A EUBACTERIUM SP. DEMONSTRATED BY THE NEGATIVE STAINING TECHNIQUE.

Bladen, Howard A. (National Institute of Dental Research, Bethesda, Md.), Marie U. Nylen, and Robert J. Fitzgerald. Internal structures of a Eubacterium sp. demonstrated by the negative staining technique. J. Bacteriol. 88:763-770. 1964.-Thin sections as well as negatively stained whole cells of a Eubacterium sp. isolated from the rat cecum were examined in a Siemens Elmiskop I electron microscope. The cell wall usually appeared in thin sections as a single dense layer about 130 A thick; however, occasionally it was demonstrated to be composed of three layers. The plasma membrane was approximately 130 A wide, and was composed of a denselight-dense arrangement. Intracytoplasmic membranous elements continuous with the plasma membrane were also observed in thin sections. When whole cells were negatively stained with phosphotungstic acid, the cell wall became transparent, and structures comparable in morphology to intracytoplasmic membranous elements were observed. These were demonstrated by stereomicroscopy to be in the interior environment of the cell, and appeared in many cases continuous with the cell wall. Frequently, they seemed to open to the exterior of the cell through what may be termed a pore. Small stalked structures similar to those described in mitochondria were observed along the periphery of the cell and occasionally along the walls of the internal elements.

Cecum↗
PubMed1988

On the fluorometric assay of circulating lipoperoxides.

The methodology for the measurement of circulating thiobarbituric acid (TBA)-reactive products of lipid peroxidation in human plasma by means of fluorometry has been reinvestigated. The lipid precipitation of plasma with phosphotungstic acid, which step is laid down in the standard assay method, strongly increases the apparent TBA-reactivity. However, subsequent washing of the precipitate may reduce the apparent levels of lipoperoxide to values close to those obtained by using untreated plasma. Also, short-time storage of plasma at either 4 degrees C or -20 degrees C induces large day to day variations in assay results. This phenomenon may be prevented almost completely by the addition of glutathione plus EDTA. It is proposed to use untreated plasma and to perform the assay immediately after collection of plasma or to add both glutathione and EDTA before storage.

Edetic Acid↗
PubMed1965

FINE STRUCTURE OF THE COAT AND NUCLEOID MATERIAL OF FOWLPOX VIRUS.

Hyde, James M. (University of Mississippi School of Medicine, Jackson), Lanelle G. Gafford, and Charles C. Randall. Fine structure of the coat and nucleoid material of fowlpox virus. J. Bacteriol. 89:1557-1569. 1965.-Several morphological forms characteristic of the poxvirus group were demonstrated for fowlpox virus with neutral phosphotungstic acid (PTA). Viral particles (purified from viral inclusion bodies) stained with uranyl acetate (UA) and shadowed with platinum were shown to have an external knobby surface not evident with PTA. The external coat of freshly purified viral particles seemed intact, but as the preparation aged, it appeared to unwind, resulting in twisted "rope-like" structures. This process was facilitated by use of 1% trypsin, and three dense fibrils were identified with UA within the partially detached viral coat. Studies with alkaline PTA (pH 9) were interpreted as revealing a complex nucleoid, but solutions above this pH damaged the particles. The morphology of the nucleoid was better depicted in ultrathin sections of whole virus which, when stained with UA, revealed dense coiled threads. Treatment of virus with sodium lauryl sulfate exposed an underlying coat consisting of small subunits approximately 40 A in diameter. Of great interest was the demonstration that the detergent removed strands of deoxyribonucleic acid (DNA) from the virus without destroying the contour of the particle. The origin of the strands was definitely the fine uranophilic, coiled threads of the nucleoid, which probably represent the DNA molecule(s). That the extracted material was largely DNA was proved by digestion with deoxyribonuclease and resistance to ribonuclease and trypsin. These studies illustrate how a variety of electron microscopic techniques may be utilized alone or in combination to reveal hitherto undescribed fine structure of viral particles.

Animals↗