Yerach Daskal

Preliminary Observations of the Effects on Breast Adenocarcinoma of Plasma Perfused over Immobilized Protein A

PROTEIN A, a constituent of the cell wall of Staphylococcus aureus Cowans 1 (SpA), reacts with the Fc region of immunoglobulins from many mammalian species and combines with immune complexes in ser...

Evidence from studies on segregated nucleoli that nucleolar silver staining proteins C23 and B23 are in the fibrillar component

Abstract Several procedures for the silver staining of nucleoli have been evaluated at the electron microscopic level to determine optimal conditions for ultrastructural preservation and staining specificity. The present study shows that a brief fixation with 1% buffered formaldehyde followed by methanol: acetic acid (3 : 1) fixation yielded optimal preservation and silver staining of nucleoli. Using this procedure for electron microscopic studies of interphase nucleoli, it was found that the punctate silver grains observed by light microscopy were composed of fine silver granules, of approx. 100 A diameter, organized in discrete clusters. In similar studies on adriamycin-induced segregated nucleoli, it was observed that the silver staining reaction was mainly limited to the fibrillar portion of the nucleolus. Accordingly, nucleolar proteins C23 and B23, found earlier to be the major silver binding proteins of the nucleolus, are mainly concentrated in the fibrillar nucleolar component.

Scanning-electron microscopy of chromosome aberrations.

This study is the first report of scanning-electron microscopy of isolated and purified metaphase chromosomes containing drug-induced aberrations. The technique reported allows high resolution topological examination of chromosomal aberrations which may pass undetected with conventional techniques.

A reciprocal relationship between contents of free ubiquitin and protein A24, its conjugate with histone 2A, in chromatin fractions obtained by the DNase II, Mg++ procedure.

Abstract Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage ( Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA 74 , 864–868 ). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H 2 SO 4 soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH 2 -terminal amino acid and its amino acid composition.

The effect of diuretic pre-treatment on clinical, morphological and ultrastructural cis-platinum induced nephrotoxicity

Abstract The clinical, microscopical and ultrastructural effects of cis -platinum induced nephrotoxicity and the effects of pre-treatment with mannitol, furosemide (Lasix) or both diuretics in male Holtzman rats is described. Cis -platinum administered intraperitoneally at doses ranging from 2.5–7.5 mg/kg was associated with dose-dependent BUN and creatinine elevation; however, weight loss, which averaged 13%, was not dose dependent. Mannitol (4 g/kg) administered immediately prior to cis -platinum, or furosemide (2 or 12.5 mg/kg) administered either concomitantly or 30 minutes prior to cis -platinum, had no effect on BUN or creatinine elevations. Varying the route or time of furosemide administration did not alter the severity of nephrotoxicity but higher doses of furosemide were associated with more severe BUN elevation. Light microscopy of renal tissue demonstrated that the principle lesion corresponding to cis-platinum injury consisted of coagulative necrosis of tubular epithelium at the corticomedullary transition zone. Glomeruli showed no observable injury. The electron microscopy confirmed the observations from light microscopy, showing injured proximal tubular cells, with loss of the characteristic nuclear electron density, segregated nucleoli and cytoplasmic degeneration. The basement membrane of the proximal tubule, the distal tubular cells and the glomeruli were perserved. Analogous to the nephrotoxicity of aminoglycoside antibiotics, diuretic treatment of animals receiving cis -platinum aggravates rather than ameliorates nephrotoxicity.

Ultrastructural and biochemical studies of the isolated fibrillar component of nucleoli from Novikoff hepatoma ascites cells

Abstract Nucleoli of Novikoff hepatoma ascites cells were fractionated into granular and fibrillar components with solutions containing polyvinsulphate and Mg 2+ (PVS/Mg 2+ ) or solutions containing EDTA. After treatment with the PVS/Mg 2+ solutions, the morphology of the fibrillar component was similar to that in situ inasmuch as they contain fibrillar clusters as well as other fibrillar elements ranging in diameter from 150–700 A. The nucleotide composition of the RNA of the fibrillar and granular fractions was similar to that of ribosomal RNA. In agreement with earlier autoradiographic studies, the kinetics of incorporation of 32 P-orthophosphate into RNA in both fractions showed that the specific activity of the RNA of the fibrillar fraction was fourfold greater than that of RNA in the granular region at 15 min. After 2 h the specific activity of the RNA in the granular region was 1.5 times greater than that in the fibrillar region. Two-dimensional polyacrylamide gel electrophoresis showed that the fibrillar fraction contained fewer proteins than the granular fraction. Some of the proteins were common to both the nucleolar granular particles and cytoplasmic ribosomes while others appeared to be unique to this fraction.

Use of direct current sputtering for improved visualization of chromosome topology by scanning electron microscopy.

Abstract The topological features of isolated Chinese hamster ovary metaphase chromosomes were studied with high resolution scanning electron microscopy (SEM) using the techniques of direct current sputtering for the deposition of metal on the specimens. Metaphase chromosome surfaces consist of numerous compact microconvules of an average diameter of 520 ± 78 A when corrected for the thickness of the gold-palladium coating (80 ± 2 A). These microconvules contain several orders of supercoiling. The superhelical structures were detected also in water-spread preparations. Most of the isolated chromosomes had membrane-like structures attached at the distal portions of the chromatids forming a terminal “plate”. Limited tryptic digests of such isolated chromosomes resulted in considerable stretching of the chromatids and revealed a series of interchromatidal fibers with diameters of 203 ± 38 A (corrected for gold coating). Treatment of these chromosomes with EDTA revealed a longitudinal array of fibers within the chromatids. The diameters of these fibers decreased as the concentration of EDTA was increased. The technique of direct current sputtering for the preparation of chromosomes for scanning microscopy is satisfactory for detailed topological ultrastructural studies in the 70 A range.

Chapter 1 Methods for Isolation of Nuclei and Nucleoli

Publisher Summary Procedures for the isolation of nuclei are important in studies on histones and other nuclear proteins, nuclear enzymes, and nuclear RNA and DNA. The objectives of procedures for isolation of nuclei are to secure a product where (1) the nuclei are morphologically identical to those of the whole cell, (2) the contents of the nuclei as they exist in the cell are all present in the isolated product, and (3) the isolated nuclei do not contain cytoplasmic constituents. The basis for the compression-decompression procedure for isolation of nucleoli is that such delicate structures as chromosomes can be obtained from nuclei without serious damage to their morphology by alternate aspiration and ejection of nuclear suspensions from a syringe. Whereas, the basis of the chemical technique for preparation of the nucleolar fraction is successive extraction of the nuclei with dilute and concentrated saline solution. The chapter also outlines the sucrose–calcium procedure for isolation of nuclei, the citric acid procedure for isolation of nuclei, the use of detergents for nuclear isolation, and the sonication procedure for isolation of nucleoli.

Isolation and partial characterization of perichromatin granules: A unique class of nuclear RNP particles

Abstract Perichromatin granules (PCG) have been isolated from cycloheximide-treated rat liver nuclei by a procedure that preserved their ultrastructural characteristics. Like the PCG particles in situ, the isolated granules were 300–400 A in diameter; they had an approximate sedimentation coefficient of 40S. The Bernhard bleaching procedure showed that the isolated perichromatin granules are not chromatinous components. A low molecular weight 4.7S RNA approx. 100 nucleotides long was associated with the granules. Analysis of the proteins of the isolated perichromatin granules on SDS polyacrylamide gel electrophoresis showed one major polypeptide (mol. wt approx. 34 000) along with two other minor polypeptides (mol. wt 31 000 and 38 000). The major polypeptide found in the perichromatin granules had similar migration characteristics on SDS gels to a peptide found in both rat liver and HeLa cell heteronuclear ribonucleoprotein (hnRNP) particles.

Ultrastructural and biochemical studies on ribonucleoprotein particles from isolated nucleoli of thioacetamide-treated rat liver.

Abstract The morphology of isolated nucleolar ribonucleoprotein particles from thioacetamide-treated rat livers was found to be very similar to those in situ. The sedimentation profiles of these nucleolar ribonucleoprotein particles in sucrose density gradients showed the presence of three components. The particles in these peaks were electron opaque spherical particles that were quite homogeneous in size (200–250 A). The ultrastructure of these RNP particles from thioacetamide-treated livers is similar to that of both ribosomes and intranucleolar RNP particles inasmuch as at high magnifications a convoluted, linear strand of RNA was observed to be present in each of the particles. In each peak of the sedimentation profile, the average diameter of the RNP particles was also 210 A. The diameters of the nucleolar granules in situ were essentially the same as those of the isolated ribonucleoprotein particles averaging 210 A and ranging from 190–250 A. The RNA in the isolated ribonucleoprotein particles was mainly 28S RNA. Quantitative analysis indicates that the RNP particles in the most rapidly sedimenting RNP peak had a higher RNA to protein ratio than those in the less rapidly sedimenting peaks.