scholarly journals Interaction of aldolase with actin-containing filaments. Structural studies

1980 ◽  
Vol 186 (1) ◽  
pp. 99-104 ◽  
Author(s):  
M Stewart ◽  
D J Morton ◽  
F M Clarke
Keyword(s):  
Structural Changes ◽  
Preceding Paper ◽  
Ultrastructural Changes ◽  
Cross Linking ◽  
Fructose Bisphosphate ◽  
Major Band ◽  
Minor Band ◽  
Computer Methods ◽  
Fructose Bisphosphate Aldolase ◽  
A Minor

Electron micrographs of the paracrystals formed when fructose bisphosphate aldolase (EC 4.1.2.13) is added to actin-containing filaments were analysed by computer methods so that ultrastructural changes could be correlated with the various stoicheiometries of binding determined in the preceding paper [Walsh, Winzor, Clarke, Masters & Morton (1980) Biochem. J. 186, 89-98]. Paracrystals formed with aldolase and either F-actin or F-actin-tropomyosin have a single light transverse band every 38 nm, which is due to aldolase molecules cross-linking the filaments. In contrast, the paracrystals formed between aldolase and F-actin-tropomyosin-troponin filaments show two transverse bands every 38 nm: a major band, interpreted as aldolase binding to troponin, and a minor band, interpreted as aldolase cross-linking the filaments. The intensity of the minor band varies with Ca2+ concentration, being greatest when the Ca2+ concentration is low. A model for the different paracrystal structures which relates the various patterns and binding stoicheiometries to structural changes in the actin-containing filaments is proposed.

scholarly journals The dominant PNM2- mutation which eliminates the psi factor of Saccharomyces cerevisiae is the result of a missense mutation in the SUP35 gene.

Genetics ◽  
1994 ◽  
Vol 137 (3) ◽  
pp. 659-670 ◽  
Author(s):  
S M Doel ◽  
S J McCready ◽  
C R Nierras ◽  
B S Cox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Suppressor Gene ◽  
Chain Termination ◽  
Major Band ◽  
Minor Band ◽  
Terminal Domain ◽  
Translation Fidelity ◽  
Extrachromosomal Element ◽  
A Minor ◽  
Sup35 Gene

Abstract The PNM2- mutation of Saccharomyces cerevisiae eliminates the extrachromosomal element psi. PNM2 is closely linked to the omnipotent suppressor gene SUP35 (also previously identified as SUP2, SUF12, SAL3 and GST1). We cloned PNM2- and showed that PNM2 and SUP35 are the same gene. We sequenced the PNM2- mutant allele and found a single G-->A transition within the N-terminal domain of the protein. We tested the effects of various constructs of SUP35 and PNM2- on psi inheritance and on allosuppressor and antisuppressor functions of the gene. We found that the C-terminal domain of SUP35 protein (SUP35p) could be independently expressed; expression produced dominant antisuppression. Disruption of the N-terminal domain of PNM2- destroyed the ability to eliminate psi. These results imply that the domains of SUP35p act in an antagonistic manner: the N-terminal domain decreases chain-termination fidelity, while the C-terminal domain imposes fidelity. Two transcripts were observed for SUP35, a major band at 2.4 kb and a minor band at 1.3 kb; the minor band corresponds to 3' sequences only. We propose a model for the function of SUP35, in which comparative levels of N- and C-terminal domains of SUP35p at the ribosome modulate translation fidelity.

Erythrina Cristagalli Lectin-reactive α-fetoprotein-E2: A Marker of Hepatocellular Carcinoma and Other Malignancies

1998 ◽  
Vol 13 (1) ◽  
pp. 24-29 ◽  
Author(s):  
M. Kamei ◽  
A. Misawa ◽  
J. Arai ◽  
K. Kamakura ◽  
K. Taketa
Keyword(s):  
Hepatocellular Carcinoma ◽  
Chronic Hepatitis ◽  
Yolk Sac ◽  
Antibody Affinity ◽  
Major Band ◽  
Minor Band ◽  
Affinity Electrophoresis ◽  
Hepatocellular Carcinomas ◽  
Yolk Sac Tumors ◽  
A Minor

A newly isolated lectin Erythrina cristagalli (ECL) was tested for separation of human α-fetoprotein (AFP) glycoforms by affinity electrophoresis at 0.5 mg/ml and separated AFP bands were detected by antibody-affinity blotting. Three AFP bands, AFP-E1, AFP-E2 and AFP-E3 in order of increasing affinity, were obtained. Sera from control patients with chronic hepatitis and cirrhosis gave a major band of AFP-E1 and a minor or trace band of AFP-E2 (3.4±2.3%), while those from patients with mostly advanced hepatocellular carcinomas had increased proportions of AFP-E2 band (16.6±10.2%). With a cutoff level of 8% (mean+2SD of AFP-E2 for controls), the sensitivity for hepatocellular carcinoma was 72% at a specificity of 100%. Gastrointestinal tumors had much higher percentages of AFP-E2 and occasionally positive AFP-E3. Most of the yolk sac tumors examined showed AFP-E3 in addition to AFP-E2, although AFP-E3 was a minor band. Thus, AFP-E2 is potentially a clinically useful marker for differentiation of increased AFP in hepatocellular carcinoma and other malignancies from that in precancerous chronic hepatitis or cirrhosis.

scholarly journals Reduction of Sulphite to Sulphide Catalysed by Desulfoviridin From Desulfovibrio gigas

1974 ◽  
Vol 27 (1) ◽  
pp. 7 ◽  
Author(s):  
HE Jones ◽  
GW Skyring
Keyword(s):  
Column Chromatography ◽  
Gel Electrophoresis ◽  
Major Band ◽  
Minor Band ◽  
Desulfovibrio Gigas ◽  
A Minor

Desulfoviridin from D. gigas was partially purified by column chromatography and further purified by gel electrophoresis into a major band preparation and a minor band preparation. All partially purified and electrophoretically purified preparations catalysed the methylviologen-linked reduction of sulphite to sulphide, but stoichiometric reduction to sulphide was not demonstrated with the major band preparation. Desulfoviridin did not catalyse the reduction of thiosulphate or trithionate.

scholarly journals The purification and properties of human liver ketohexokinase. A role for ketohexokinase and fructose-bisphosphate aldolase in the metabolic production of oxalate from xylitol

1985 ◽  
Vol 230 (1) ◽  
pp. 53-60 ◽  
Author(s):  
R Bais ◽  
H M James ◽  
A M Rofe ◽  
R A Conyers
Keyword(s):  
Human Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Kinetic Properties ◽  
Fructose Bisphosphate ◽  
Reaction Sequence ◽  
Purification And Properties ◽  
Phosphorylated Compounds ◽  
Fructose Bisphosphate Aldolase ◽  
A Minor ◽  
Inhibition Studies

Ketohexokinase (EC 2.7.1.3) was purified to homogeneity from human liver, and fructose-bisphosphate aldolase (EC 4.1.2.13) was partially purified from the same source. Ketohexokinase was shown, by column chromatography and polyacrylamide-gel electrophoresis, to be a dimer of Mr 75000. Inhibition studies with p-chloromercuribenzoate and N-ethylmaleimide indicate that ketohexokinase contains thiol groups, which are required for full activity. With D-xylulose as substrate, ketohexokinase and aldolase can catalyse a reaction sequence which forms glycolaldehyde, a known precursor of oxalate. The distribution of both enzymes in human tissues indicates that this reaction sequence occurs mainly in the liver, to a lesser extent in the kidney, and very little in heart, brain and muscle. The kinetic properties of ketohexokinase show that this enzyme can phosphorylate D-xylulose as readily as D-fructose, except that higher concentrations of D-xylulose are required. The kinetic properties of aldolase show that the enzyme has a higher affinity for D-xylulose 1-phosphate than for D-fructose 1-phosphate. These findings support a role for ketohexokinase and aldolase in the formation of glycolaldehyde. The effect of various metabolites on the activity of the two enzymes was tested to determine the conditions that favour the formation of glycolaldehyde from xylitol. The results indicate that few of these metabolites affect the activity of ketohexokinase, but that aldolase can be inhibited by several phosphorylated compounds. This work suggests that, although the formation of oxalate from xylitol is normally a minor pathway, under certain conditions of increased xylitol metabolism oxalate production can become significant and may result in oxalosis.

Fine Structural Changes in the Microvasculature of the Mouse Pinna: Aging and Radiation Injury

1974 ◽  
Vol 32 ◽  
pp. 54-55
Author(s):  
S. Phyllis Steamer ◽  
Rosemarie L. Devine
Keyword(s):  
Structural Changes ◽  
Radiation Treatment ◽  
Arterial Stenosis ◽  
Ultrastructural Changes ◽  
Vascular Effects ◽  
Microvascular Changes ◽  
Surrounding Connective Tissue ◽  
Fission Neutrons ◽  
Total Body

The importance of radiation damage to the skin and its vasculature was recognized by the early radiologists. In more recent studies, vascular effects were shown to involve the endothelium as well as the surrounding connective tissue. Microvascular changes in the mouse pinna were studied in vivo and recorded photographically over a period of 12-18 months. Radiation treatment at 110 days of age was total body exposure to either 240 rad fission neutrons or 855 rad 60Co gamma rays. After in vivo observations in control and irradiated mice, animals were sacrificed for examination of changes in vascular fine structure. Vessels were selected from regions of specific interest that had been identified on photomicrographs. Prominent ultrastructural changes can be attributed to aging as well as to radiation treatment. Of principal concern were determinations of ultrastructural changes associated with venous dilatations, segmental arterial stenosis and tortuosities of both veins and arteries, effects that had been identified on the basis of light microscopic observations. Tortuosities and irregularly dilated vein segments were related to both aging and radiation changes but arterial stenosis was observed only in irradiated animals.

Embedding Procedure for Water Swollen Samples

1970 ◽  
Vol 28 ◽  
pp. 504-505
Author(s):  
Ann M. Thomas ◽  
Virginia Shemeley
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Ion Exchange ◽  
Structural Changes ◽  
Cross Linking ◽  
Ion Exchange Resins ◽  
Transmission Electron ◽  
First Pass ◽  
Exchange Resins

Those samples which swell rapidly when exposed to water are, at best, difficult to section for transmission electron microscopy. Some materials literally burst out of the embedding block with the first pass by the knife, and even the most rapid cutting cycle produces sections of limited value. Many ion exchange resins swell in water; some undergo irreversible structural changes when dried. We developed our embedding procedure to handle this type of sample, but it should be applicable to many materials that present similar sectioning difficulties.The purpose of our embedding procedure is to build up a cross-linking network throughout the sample, while it is in a water swollen state. Our procedure was suggested to us by the work of Rosenberg, where he mentioned the formation of a tridimensional structure by the polymerization of the GMA biproduct, triglycol dimethacrylate.

Effects of Myo-inositol Alone and in Combination with Seleno-Lmethionine on Cadmium-Induced Testicular Damage in Mice

2019 ◽  
Vol 12 (4) ◽  
pp. 311-323 ◽  
Author(s):  
Salvatore Benvenga ◽  
Antonio Micali ◽  
Giovanni Pallio ◽  
Roberto Vita ◽  
Consuelo Malta ◽  
...  
Keyword(s):  
Structural Changes ◽  
Natural Antioxidants ◽  
Minor Role ◽  
Positive Role ◽  
Testosterone Levels ◽  
Tnf Α ◽  
Testicular Lesions ◽  
A Minor ◽  
Immunohistochemical Analyses

Background: Cadmium (Cd) impairs gametogenesis and damages the blood-testis barrier. Objective: As the primary mechanism of Cd-induced damage is oxidative stress, the effects of two natural antioxidants, myo-inositol (MI) and seleno-L-methionine (Se), were evaluated in mice testes. Methods: Eighty-four male C57 BL/6J mice were divided into twelve groups: 0.9% NaCl (vehicle; 1 ml/kg/day i.p.); Se (0.2 mg/kg/day per os); Se (0.4 mg/kg/day per os); MI (360 mg/kg/day per os); MI plus Se (0.2 mg/kg/day); MI plus Se (0.4 mg/kg/day); CdCl2 (2 mg/kg/day i.p.) plus vehicle; CdCl2 plus MI; CdCl2 plus Se (0.2 mg/kg/day); CdCl2 plus Se (0.4 mg/kg/day); CdCl2 plus MI plus Se (0.2 mg/kg/day); and CdCl2 plus MI plus Se (0.4 mg/kg/day). After 14 days, testes were processed for biochemical, structural and immunohistochemical analyses. Results: CdCl2 increased iNOS and TNF-α expression and Malondialdehyde (MDA) levels, lowered glutathione (GSH) and testosterone, induced testicular lesions, and almost eliminated claudin-11 immunoreactivity. Se administration at 0.2 or 0.4 mg/kg significantly reduced iNOS and TNF-α expression, maintained GSH, MDA and testosterone levels, structural changes and low claudin-11 immunoreactivity. MI alone or associated with Se at 0.2 or 0.4 mg/kg significantly reduced iNOS and TNF-α expression and MDA levels, increased GSH and testosterone levels, ameliorated structural organization and increased claudin-11 patches number. Conclusion: We demonstrated a protective effect of MI, a minor role of Se and an evident positive role of the association between MI and Se on Cd-induced damages of the testis. MI alone or associated with Se might protect testes in subjects exposed to toxicants, at least to those with behavior similar to Cd.

scholarly journals Establishment of an in vitro model for analyzing mitochondrial ultrastructure in PRKN-mutated patient iPSC-derived dopaminergic neurons

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Mutsumi Yokota ◽  
Soichiro Kakuta ◽  
Takahiro Shiga ◽  
Kei-ichi Ishikawa ◽  
Hideyuki Okano ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Dopaminergic Neurons ◽  
Structural Changes ◽  
In Vitro Model ◽  
Induced Pluripotent Stem Cell ◽  
Substantia Nigra Pars Compacta ◽  
Ultrastructural Changes ◽  
Mitochondrial Morphology ◽  
Vitro Model

AbstractMitochondrial structural changes are associated with the regulation of mitochondrial function, apoptosis, and neurodegenerative diseases. PRKN is known to be involved with various mechanisms of mitochondrial quality control including mitochondrial structural changes. Parkinson’s disease (PD) with PRKN mutations is characterized by the preferential degeneration of dopaminergic neurons in the substantia nigra pars compacta, which has been suggested to result from the accumulation of damaged mitochondria. However, ultrastructural changes of mitochondria specifically in dopaminergic neurons derived from iPSC have rarely been analyzed. The main reason for this would be that the dopaminergic neurons cannot be distinguished directly among a mixture of iPSC-derived differentiated cells under electron microscopy. To selectively label dopaminergic neurons and analyze mitochondrial morphology at the ultrastructural level, we generated control and PRKN-mutated patient tyrosine hydroxylase reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines. Correlative light-electron microscopy analysis and live cell imaging of GFP-expressing dopaminergic neurons indicated that iPSC-derived dopaminergic neurons had smaller and less functional mitochondria than those in non-dopaminergic neurons. Furthermore, the formation of spheroid-shaped mitochondria, which was induced in control dopaminergic neurons by a mitochondrial uncoupler, was inhibited in the PRKN-mutated dopaminergic neurons. These results indicate that our established TH-GFP iPSC lines are useful for characterizing mitochondrial morphology, such as spheroid-shaped mitochondria, in dopaminergic neurons among a mixture of various cell types. Our in vitro model would provide insights into the vulnerability of dopaminergic neurons and the processes leading to the preferential loss of dopaminergic neurons in patients with PRKN mutations.

scholarly journals Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents

1988 ◽  
Vol 255 (3) ◽  
pp. 869-876 ◽  
Author(s):  
D J Steenkamp
Keyword(s):  
Electron Transfer ◽  
Small Subunit ◽  
Cross Linking ◽  
Minor Effect ◽  
Electron Transfer Flavoprotein ◽  
Ubiquinone Oxidoreductase ◽  
Lysine Residues ◽  
Cross Links ◽  
A Minor ◽  
Chemical Cross Linking

The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2′-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.

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