Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes

1975 ◽  
Vol 17 (1) ◽  
pp. 113-139
Author(s):  
M.E. Walmsley ◽  
H.G. Davies
Keyword(s):  
Structural Changes ◽  
Intact Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Condensed Chromatin ◽  
Isolation Medium ◽  
Thin Sections ◽  
Shell Forming ◽  
Chicken Erythrocytes ◽  
O Phase

Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium ions (1 mM), sucrose (0-4 M) and Tris buffer (0.01 M, pH 7-5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and e-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The 0-phase is also largely retained after extensive washing in 0-07 M NaC1 as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%. These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28nm. The 5–10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely fibrillar body.

Electron-Microscope Observations on the Organization of the Nucleus in Chicken Erythrocytes and a Superunit Thread Hypothesis for Chromosome Structure

1974 ◽  
Vol 16 (2) ◽  
pp. 261-299 ◽  
Author(s):  
H. G. DAVIES ◽  
A. B. MURRAY ◽  
M. E. WALMSLEY
Keyword(s):  
Electron Microscope ◽  
Central Element ◽  
Condensed Chromatin ◽  
Structural Units ◽  
Protein Ratio ◽  
Intact Cells ◽  
Thin Sections ◽  
Shell Forming ◽  
Chicken Erythrocytes ◽  
O Phase

Previously it was shown that when condensed chromatin from several different types of cell is stained with uranyl-lead and examined in thin sections in the electron microscope, the stain is distributed into a dot-dash pattern arising from threads, with lesser-staining intermediate areas. We now show that when a section through chicken erythrocyte chromatin is stained with ethanolic phosphotungstic acid (PTA) the stain distribution is homogeneous. This shows that the lesser-staining regions after uranyl-lead, cannot be an overlap artifact. We conclude that the stains and hence the molecules in chromatin are distributed between 2 phases, an o- and an e-phase, so called because the structural units in chromatin are arranged in an orderly way at the surface of the nucleus and give rise to oddly (o) and evenly (e) numbered bands. Measurements of electron density per unit thickness, proportional to the number of stain molecules per unit volume, are made in thin sections through erythrocytes and reticulocytes from adult hen, 4-day-old chicks and 17-day embryos. The results indicate differences in the packing of the molecules in chromatin and further show that the e-phase is quite likely to have a higher DNA to protein ratio than the o-phase. After uranyl-lead stain the visibility of the dot-dash pattern in cells from adult hen is relatively low due, we propose, to closer packing. In micrographs through condensed chromatin treated with uranyl-lead the eye selects out only the densely stained dots and dashes, width 17 nm. When erythrocyte chromatin is partially or completely disrupted in various ways, threads 25-30 nm then become visible. We propose that condensed chromatin in intact cells contains structural units which consist of a central element, width 17 nm previously referred to as the unit thread, forming the e-phase, surrounded by a cylindrical shell forming the o-phase. This socalled superunit thread is similar in width, about 25-30 nm, to that reported by other workers in preparations of chromosomes spread on water surfaces. The hypothesis therefore helps explain what appeared to be discrepancies in thread dimensions. Certain other ultrastructural features of erythrocyte nuclei are also reported which are either pertinent to the general aim of this study, namely the way in which nucleoproteins fold up in chromosomes, or to biochemical studies, to be reported shortly, in which attempts are made to locate the proteins removed from isolated erythrocyte nuclei during subsequent washing in salt solutions.

scholarly journals Effects of High Intensity Ultrasound on Physiochemical and Structural Properties of Goat Milk β-Lactoglobulin

Molecules ◽  
2020 ◽  
Vol 25 (16) ◽  
pp. 3637
Author(s):  
Xinhui Zhou ◽  
Cuina Wang ◽  
Xiaomeng Sun ◽  
Zixuan Zhao ◽  
Mingruo Guo
Keyword(s):  
Thermal Stability ◽  
Structural Properties ◽  
High Intensity ◽  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Goat Milk ◽  
High Intensity Ultrasound ◽  
Hydrophobicity Index ◽  
Β Lactoglobulin

This study aimed to compare the effects of high intensity ultrasound (HIU) applied at various amplitudes (20~40%) and for different durations (1~10 min) on the physiochemical and structural properties of goat milk β-lactoglobulin. No significant change was observed in the protein electrophoretic patterns by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Deconvolution and second derivative of the Fourier transform infrared spectra (FTIR) showed that the percentage of β-sheet of goat milk β-lactoglobulin was significantly decreased while those of α-helix and random coils increased after HIU treatment The surface hydrophobicity index and intrinsic fluorescence intensity of samples was enhanced and increased with increasing HIU amplitude or time. Differential scanning calorimetry (DSC) results exhibited that HIU treatments improved the thermal stability of goat milk β-lactoglobulin. Transmission electron microscopy (TEM) of samples showed that the goat milk β-lactoglobulin microstructure had changed and it contained larger aggregates when compared with the untreated goat milk β-lactoglobulin sample. Data suggested that HIU treatments resulted in secondary and tertiary structural changes of goat milk β-lactoglobulin and improved its thermal stability.

Rapid detection and initial characterization of genetic variants of human serum albumin

1991 ◽  
Vol 37 (7) ◽  
pp. 1221-1224 ◽  
Author(s):  
J Merle Sheat ◽  
Robert J Peach ◽  
Peter M George
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Genetic Variants ◽  
Gel Electrophoresis ◽  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl

Abstract We have studied the detection and classification of genetic variants of human serum albumin by electrophoresis. Samples from 10 patients who were heterozygous for eight different albumin variants were studied by two methods. In agarose gel electrophoresis, each of these variants has an abnormal mobility and can be classified on the basis that structural changes at the N-terminus abolish 63Ni binding. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole serum, glycosylated variants are easily detected because of their greater apparent molecular mass.

scholarly journals Natural Occurrence of Chilli veinal mottle virus on Capsicum chinense in China

Plant Disease ◽  
2006 ◽  
Vol 90 (3) ◽  
pp. 377-377 ◽  
Author(s):  
J. Wang ◽  
Z. Liu ◽  
S. Niu ◽  
M. Peng ◽  
D. Wang ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Viral Disease ◽  
Mottle Virus ◽  
Major Protein ◽  
Natural Occurrence ◽  
Capsicum Chinense ◽  
Thin Sections ◽  
First Report ◽  
Chilli Veinal Mottle Virus

An outbreak of a viral disease on chili pepper (Capsicum chinense Jacp. cv. Yellow Lantern) occurred in Hainan Province, China during 2003 and 2004. The disease was prevalent in five chili-producing counties surveyed. Leaves of infected plants initially displayed symptoms of dark green banding along veins and later became distorted with striking mosaic. Infected plants had reduced flower numbers and fruit set, resulting in a significant yield loss. The causative virus was characterized and identified as Chilli veinal mottle virus (ChiVMV) (3). An isolate of the virus was obtained via three single lesion passages through Chenopodium amaranticolor and was shown to reproduce the same symptoms on inoculated C. chinense cv. Yellow Lantern. Negative staining of crude extracts of the infected tissue and subsequent electron microscopy revealed flexuous rods of 12 to 13 × 750 nm, typical of a potyvirus. Pinwheel-like inclusion bodies were abundant in thin sections of infected leaves. Purified virus preparations contained one major protein of 32.8 kDa and one minor protein of 28 kDa when fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both of these protein bands were excised and subsequently analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple peptide fragments from both proteins were identified as arising from ChiVMV capsid protein (CP) (1,2). Therefore, the 32.8-kDa protein is the full-length ChiVMV CP and the 28-kDa protein is presumably a degradation product of the CP. The combined biological and molecular data provided strong evidence that the viral disease on C. chinense was caused by ChiVMV. To our knowledge, this is the first report of ChiVMV infection on C. chinense in China and the first report of C. amaranticolor as an experimental host for ChiVMV. References: (1) P. Chiemsombat et al. Arch. Virol. 143:1855, 1998. (2). J. Joseph and H. S. Savithri. Arch. Virol. 144:1679, 1999. (3) P. Siriwong et al. Plant Pathol. 44:718, 1995.

scholarly journals Contribution of Chemical Modifications and Conformational Epitopes to IgE Binding by Ara h 3

Foods ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 189 ◽  
Author(s):  
Scott Dyer ◽  
Jacqueline Nesbit ◽  
Beatriz Cabanillas ◽  
Hsiaopo Cheng ◽  
Barry Hurlburt ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Secondary Structure ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cd Spectroscopy ◽  
Chemical Modifications ◽  
Ige Binding ◽  
Ara H 3

Roasting is known to change the allergenic properties of peanuts. To study these observations at a molecular level, the relationship of IgE binding to the structure of Ara h 3 from raw and roasted peanuts was assessed. Ara h 3 (A3) was purified from raw (R), light roast (LR) and dark roast (DR) peanuts, the purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the secondary structures were compared with circular dichroism (CD) spectroscopy. In order to understand the contribution of structure to IgE binding, the R A3 was partially denatured (PD) by heat treatment (65 °C for 2 h), subjected to CD spectroscopy and IgE spot blot analysis with sera from peanut- allergic individuals. While we observed that the secondary structure of purified A3 from R and LR peanut in solution was affected by the reduction of disulfide bonds and heat treatment when purified from the peanut following the roasting process, only small alterations were seen in the secondary structure. The purified LR A3 bound higher levels of IgE than the RA3. CD spectroscopy of PD A3 revealed a reduction in the percentage of alpha helices, and serum IgE binding. Therefore, while A3 purified from roasted peanuts did not show significant changes in secondary structure, it showed higher IgE binding than R A3. Therefore, the higher IgE binding to LR A3 was more likely to be due to chemical modifications than structural changes. However, a decrease in the IgE binding was seen if R A3 was deliberately unfolded, indicating that the structure played an important role in IgE binding to A3.

scholarly journals Subaxolemmal filamentous network in the giant nerve fiber of the squid (Loligo pealei L.) and its possible role in excitability.

1978 ◽  
Vol 78 (2) ◽  
pp. 597-621 ◽  
Author(s):  
J Metuzals ◽  
I Tasaki
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Nerve Fiber ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Three Dimensional ◽  
Polarized Light ◽  
Thin Sections ◽  
Loligo Pealei ◽  
Scanning Electron

A new technique utilizing the squid giant nerve fiber has been developed which permits direct examination of the inner face of the axolemma by scanning electron microscopy. The axoplasm was removed sequentially in a 15-mm long segment of the fiber by intracellular perfusion with a solution of KF, KCl, Ca++-containing seawater, or with pronase. The action potential of the fibers was monitored during these treatments. After brief prefixation in 1% paraformaldehyde and 1% glutaraldehyde, the perfused segment was opened by a lne could be related to information on the detailed morphology of the cytoplasmic face of the axolemma and the ectoplasm. The results obtained by scanning electron microscopy were further substantiated by transmission electron microscopy of thin sections. In addition, living axons were studied with polarized light during axoplasm removal, and the identification of actin by heavy meromyosin labeling and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was accomplished. These observations demonstrate that a three-dimensional network of interwoven filaments, consisting partly of an actinlike protein, is firmly attached to the axolemma. The axoplasmic face of fibers in which the filaments have been removed partially after perfusion with pronase displays smooth membranous blebs and large profiles which sppose the axolemma. In fibers where the excitability has been suppressed by pronase perfusion, approximately one-third of the inner face of the axolemma in the perfusion zone is free of filaments. It is hypothesized that the attachment of axoplasm filaments to the axolemma may have a role in the maintenance of the normal morphology of the axolemma, and, thus, in some aspect of excitability.

scholarly journals Biological and Molecular Characterization of a Novel Carmovirus Isolated from Angelonia

2006 ◽  
Vol 96 (5) ◽  
pp. 460-467 ◽  
Author(s):  
Scott Adkins ◽  
John Hammond ◽  
Abed Gera ◽  
Clarissa J. Maroon-Lango ◽  
Irena Sobolev ◽  
...  
Keyword(s):  
United States ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Particle Morphology ◽  
The United States ◽  
Open Reading Frames ◽  
Enzyme Linked Immunosorbent Assay ◽  
Major Protein ◽  
Thin Sections ◽  
Phlox Drummondii

A new carmovirus was isolated from Angelonia plants (Angelonia angustifolia), with flower break and mild foliar symptoms, grown in the United States and Israel. The virus, for which the name Angelonia flower break virus (AnFBV) is proposed, has isometric particles, ≈30 nm in diameter. The experimental host range was limited to Nicotiana species, Schizanthus pinnatus, Myosotis sylvatica, Phlox drummondii, and Digitalis purpurea. Virions were isolated from systemically infected N. benthamiana leaves, and directly from naturally infected Angelonia leaves, using typical carmovirus protocols. Koch's postulates were completed by mechanical inoculation of uninfected Angelonia seedlings with purified virions. Isometric particles were observed in leaf dips and virion preparations from both Angelonia and N. benthamiana, and in thin sections of Angelonia flower tissue by electron microscopy. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dissociated purified virus preparations, a major protein component with a molecular mass of 38 kDa was observed. Virion preparations were used to produce virus-specific polyclonal antisera in both Israel and the United States. The antisera did not react with Pelargonium flower break virus (PFBV), Carnation mottle virus (CarMV), or Saguaro cactus virus (SgCV) by either enzyme-linked immunosorbent assay or immunoblotting. In reciprocal tests, antisera against PFBV, CarMV, and SgCV reacted only with the homologous viruses. The complete nucleotide sequence of a Florida isolate of AnFBV and the coat protein (CP) gene sequences of Israeli and Maryland isolates were determined. The genomic RNA is 3,964 nucleotides and contains four open reading frames arranged in a manner typical of carmoviruses. The AnFBV CP is most closely related to PFBV, whereas the AnFBV replicase is most closely related to PFBV, CarMV, and SgCV. Particle morphology, serological properties, genome organization, and phylogenetic analysis are all consistent with assignment of AnFBV to the genus Carmovirus.

Expression, purification and structural analysis of recombinant rBdh-2His6, a spermadhesin from buck (Capra hircus) seminal plasma

2012 ◽  
Vol 24 (4) ◽  
pp. 580 ◽  
Author(s):  
Antônia Sâmia F. Nascimento ◽  
João B. Cajazeiras ◽  
Kyria S. Nascimento ◽  
Sara Monalisa S. Nogueira ◽  
Bruno L. Sousa ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Structural Changes ◽  
Polymerase Chain Reaction Amplification ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Exchange Chromatography ◽  
Capra Hircus ◽  
Secretory Proteins ◽  
Reaction Amplification ◽  
Polymerase Chain

Spermadhesins, a family of secretory proteins from the male genital tract of ungulate species, belong to the group of animal lectins. Spermadhesins have a prominent role in different aspects of fertilisation, such as spermatozoid capacitation, acrosomal stabilisation, sperm–oviduct interaction and during sperm–oocyte fusion. Proteins (spermadhesins) in buck seminal plasma were described. In the present study, bodhesin Bdh-2 cDNA present in buck seminal plasma was subcloned with the expression plasmid pTrcHis TOPO used to transform Escherichia coli Top10 One shot cells. The recombinant clones were selected by growth in 50 µg mL–1 ampicillin-containing LB broth and polymerase chain reaction amplification. Recombinant rBdh-2His6 synthesis was monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and followed by immunoblotting using monoclonal anti-His antibody. Production of rBdh-2 using low temperatures was not satisfactory. Greater production of rBdh-2 occurred with 1.5 mM isopropyl β-d-thiogalactoside after 2 h of induction. The method used to purify rBdh-2 was affinity chromatography on a His-Trap column following ion-exchange chromatography on a DEAE-Sephacel column. The secondary structure of the rBdh-2His6 was evaluated by spectral profile circular dichroism (CD). The prevalence of secondary structures like β-sheets, with fewer unfolded structures and α-helices, was confirmed. The structure of rBdh-2His6 remained stable up to 35°C. However, significant structural changes were observed at temperatures higher than 40°C related to a distortion of the CD spectrum.

Amikacin disrupts the cell envelope of Pseudomonas aeruginosa ATCC 9027

1988 ◽  
Vol 34 (1) ◽  
pp. 12-18 ◽  
Author(s):  
S. G. Walker ◽  
T. J. Beveridge
Keyword(s):  
Electron Microscopy ◽  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Cell Envelope ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Atomic Absorption Spectrophotometry ◽  
Fracture Pattern ◽  
Thin Sections ◽  
Biochemical Analyses

Amikacin, an aminoglycoside known to inhibit protein synthesis, was found to perturb the outer membrane of a sensitive Pseudomonas aeruginosa strain (ATCC 9027). This perturbation was monitored using electron microscopy and biochemical analyses. Following exposure to 20 μg amikacin/mL for 15 min, the outer membrane of exponentially growing cells lost 15% of its protein, 18% of its lipopolysaccharide, and 18% of its phosphate. Sodium dodecyl sulphate – polyacrylamide gel electrophoresis showed that the whole spectrum of outer membrane protein and lipopolysaccharide was affected. Similarly, atomic absorption spectrophotometry revealed that magnesium and calcium were also lost. When cells were treated with amikacin, electron microscopy of negative stains showed a substantial increase in outer membrane blebbing. Freeze fractures revealed changes in membrane fracture pattern and particle distribution, and thin sections revealed a sequential disruption of the cell envelope beginning at the outer membrane and ending at the plasma membrane. This study supports the proposal that aminoglycoside antibiotics cross the outer membrane of Pseudomonas aeruginosa by displacing metal cations necessary to stabilize the organic constituents of the membrane. Their removal results in loss of the outer membrane and the formation of transient small holes which permit the antibiotic access to the cytoplasmic membrane where it is transported into the cytoplasm.

scholarly journals Heat-Induced Interactions between Whey Protein and Inulin and Changes in Physicochemical and Antioxidative Properties of the Complexes

2019 ◽  
Vol 20 (17) ◽  
pp. 4089
Author(s):  
Wang ◽  
Wang ◽  
Sun ◽  
Sun ◽  
Guo
Keyword(s):  
Whey Protein ◽  
Structural Changes ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Lower Surface ◽  
Surface Hydrophobicity ◽  
Antioxidative Properties ◽  
Dpph Scavenging ◽  
Far Ultraviolet

Whey protein and inulin at various weight ratios were dry heated at 60 °C for 5 days under relative humidity of 63%. The heated mixtures were found to have significant changes in browning intensity and zeta-potential compared to untreated mixture. Heated samples showed significantly lower surface hydrophobicity than untreated mixtures. Compared with untreated samples, dry-heated samples showed significantly higher 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) scavenging ability with whey protein to inulin mass ratios of 1:2 and 1:3 and significantly higher 2,2′-Azinobis(2-Ethylbenzothiazoline-6-Sulfonate) (ABTS) scavenging abilities and oxygen radical absorbance capacity (ORAC) at all weight ratios. Dry heat-induced interactions between whey protein and inulin was confirmed by changes in Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profile, Fourier Transform Infrared Spectroscopy (FT-IR) and Far-ultraviolet Circular Dichroism (Far-UV CD) spectra. Dry heating caused physicochemical and structural changes of whey protein and therefore the complexes can be used to improve the antioxidative properties of the mixture under certain conditions.

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