scholarly journals Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly.

1981 ◽  
Vol 89 (3) ◽  
pp. 560-567 ◽  
Author(s):  
H M Moon ◽  
T Wisniewski ◽  
P Merz ◽  
J De Martini ◽  
H M Wisniewski
Keyword(s):  
Column Chromatography ◽  
High Speed ◽  
Bovine Brain ◽  
Urea Solution ◽  
Partial Purification ◽  
Filament Formation ◽  
Globular Structure ◽  
Backbone Structure ◽  
Cellulose Column

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high-speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.

Tryptophan 2,3-dioxygenase in Saccharomyces cerevisiae

1995 ◽  
Vol 41 (1) ◽  
pp. 19-26 ◽  
Author(s):  
Yoshiki Iwamoto ◽  
In Sook Matsui Lee ◽  
Ryo Kido ◽  
Motonari Tsubaki
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Column Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ring Cleavage ◽  
Partial Purification ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cleavage Enzyme ◽  
A Subunit ◽  
Exogenous Ligands ◽  
Cellulose Column

The tryptophan pyrrole-ring cleavage enzyme (TPCE) was detected in the yeast Saccharomyces cerevisiae. TPCE activity existed constitutively and was markedly induced by culturing the cells in a medium containing 0.1% (w/v) L-tryptophan. We purified partially the enzyme from the L-tryptophan-induced cells by phospho-cellulose column chromatography. The partially purified enzyme was stimulated solely by L-ascorbic acid, a nonspecific reductant, suggesting that the yeast TPCE is not indoleamine 2,3-dioxygenase, but rather tryptophan 2,3-dioxygenase. The enzyme metabolized L-tryptophan preferentially, and D-tryptophan slightly. KCN and NaN3, exogenous ligands of heme, inhibited the enzyme activity drastically, indicating that yeast tryptophan 2,3-dioxygenase contains heme(s) in its active site. The optimal pH of the enzyme was 6.5. Upon two-dimensional polyacrylamide gel electrophoresis, a protein staining spot was identified that was induced by L-tryptophan and whose intensity changed in correlation with the tryptophan 2,3-dioxygenase activity after phospho-cellulose column chromatography. This protein, exhibiting a molecular weight of approximately 38 000 and an isoelectric point of approximately pH 8.0, may be identified as a subunit of yeast tryptophan 2,3-dioxygenase.Key words: tryptophan 2,3-dioxygenase, Saccharomyces cerevisiae, partial purification, heme.

Chemical Analysis and Antioxidant Activities In Vitro of Polysaccharide Extracted from Corn Silk

2012 ◽  
Vol 535-537 ◽  
pp. 2335-2339 ◽  
Author(s):  
Yu Tang He ◽  
Hong Ni Gao ◽  
Yong Xia Xu ◽  
Xiao Ming Pan ◽  
Jian Rong Li
Keyword(s):  
Chemical Analysis ◽  
Hydroxyl Radical ◽  
Column Chromatography ◽  
Antioxidant Activities ◽  
Chemical Properties ◽  
Corn Silk ◽  
Scavenging Capacity ◽  
Glucosidic Bond ◽  
Cellulose Column

Corn silk polysaccharide (CSP) was investigated for the treatment of different kinds of diseases. In order to characterize the chemical properties and antioxidant activities of CSP, the CSP was isolated from corn silk and purified by DE-52 cellulose column chromatography. Four components were separated, and the highest one named CSP-A. The CSP-A was characterized by FTIR and the monosaccharide components were analyzed by HPLC. The FTIR spectra indicated that CSP-A was characteristic of β-glucosidic bond and α-glycosidic bond. The CSP-A mainly comprised of glucose, galactose, arabinose, mannose, rhamnose. The antioxidant activities of CSP were determined by hydroxyl radical (•OH) and DPPH radicals scavenging assays. When the concentration of CSP was 10mg/mL, the scavenging capacity of •OH and DPPH could reach to 40% and 48%, respectively.

The Strong Positive Correlation between Factor VII Clotting Activity Using Bovine Thromboplastin and the Activated Factor VII Level

1995 ◽  
Vol 73 (03) ◽  
pp. 429-434 ◽  
Author(s):  
Kazuomi Kario ◽  
Takefumi Matsuo ◽  
Reiko Asada ◽  
Toshiyuki Sakata ◽  
Hisao Kato ◽  
...  
Keyword(s):  
Risk Factor ◽  
Bovine Brain ◽  
Factor Vii ◽  
Rabbit Brain ◽  
Strong Positive Correlation ◽  
Chromogenic Substrate ◽  
Moderate Correlation ◽  
Positive Correlation ◽  
Activated Factor Vii

SummaryWe compared factor VII clotting activity (FVIIc) assays using different thromboplastins to determine which is the most sensitive for activated FVII (FVIIa) or for FVII antigen (FVIIag). FVIIc levels were measured using thromboplastins derived from bovine brain (FVIIc Bov), human placenta (FVIIc Hum), and rabbit brain (FVIIc Rab). FVIIa levels were measured by fluorogenic assays using human soluble tissue factor (rsTF) or bovine rsTF. We also measured FVII activity by an amidolytic assay (FVIIc:am Hum) using human thromboplastin and a chromogenic substrate for thrombin. FVIIag levels were determined by ELISA. In the FVIIa assay, the reaction time obtained from using bovine rsTF was shorter than that with human rsTF, suggesting that the interaction of plasma FVIIa with bovine rsTF was stronger than with human rsTF. The plasma FVIIa levels measured using human rsTF and bovine rsTF were almost the same (r=0.947, p<0.0001). Among the three FVIIc assays, FVIIc Bov had the strongest positive correlation with the plasma FVIIa level (r=0.886, p<0.000l), but had no correlation with FVIIag. An increase of 1 ng/ml in the plasma FVIIa level yielded a 27.9% increase of FVIIc Bov. Plasma FVIIc Hum and FVIIc:am Hum showed moderate correlations with both FVIIa (r=0.520, p<0.02 and r=0.569, p<0.01, respectively) and FVIIag (r=0.438, p<0.05 and r=0.468, p<0.05, respectively). FVIIc Rab had the lowest correlation with FVIIa (r=0.367, p<0.1), but had a moderate correlation with FVIIag (r=0.436, p<0.05). After in vitro cold activation, FVIIc Bov levels increased the most and FVIIc:am levels showed the least change. These findings indicate that consideration of the thromboplastin used for assay is necessary when assessing the clinical significance of FVII activity as a cardiovascular risk factor.

scholarly journals Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

2006 ◽  
Vol 26 (17) ◽  
pp. 6425-6434 ◽  
Author(s):  
O. Jameel Shah ◽  
Tony Hunter
Keyword(s):  
Cell Culture ◽  
Tuberous Sclerosis ◽  
High Speed ◽  
Serine Phosphorylation ◽  
Feedback Signal ◽  
Insulin Receptor Substrates ◽  
Igf I ◽  
Culture Models ◽  
Cell Culture Models

ABSTRACT The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

Inhibition of binding of gonadal steroids to serum binding proteins by non-esterified fatty acids: the influence of chain length and degree of unsaturation

1989 ◽  
Vol 120 (2) ◽  
pp. 175-179 ◽  
Author(s):  
C. Street ◽  
R. J. S. Howell ◽  
L. Perry ◽  
S. Al-Othman ◽  
T. Chard
Keyword(s):  
Fatty Acids ◽  
Protein Binding ◽  
Unsaturated Fatty Acids ◽  
Late Pregnancy ◽  
Sex Hormone Binding Globulin ◽  
Partial Purification ◽  
Normal Female ◽  
Esterified Fatty Acids ◽  
Vitro Binding

Abstract. The effect of non-esterified fatty acids (NEFA) on the in vitro binding of testosterone, 5-alpha dihydrotestosterone and estradiol E2 to sex hormone binding globulin (SHBG) was examined using pooled normal female serum, and SHBG and albumin fractions obtained from the partial purification of late pregnancy serum. A range of saturated and unsaturated fatty acids were examined for their effect on steroid-protein binding. In normal female serum, NEFA added at physiological concentrations disrupted steroid-protein binding. The shorter chain (C8–C12) saturated acids and the poly-unsaturated acids proved to be more effective inhibitors than the longer chain saturated or mono-unsaturated acids. The greatest inhibition was obtained with E2 whereas the binding of dihydrotestosterone was least affected. With partially purified SHBG, the same concentrations of NEFA were less effective at inhibiting the binding of dihydrotestosterone and testosterone but elicited the same effect with E2. The binding of steroids to albumin appeared to be unaffected by these concentrations of NEFA.

A simple assay for galactokinase using deae-cellulose column chromatography

1977 ◽  
Vol 74 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Y.S. Shin-Buehring ◽  
M. Osang ◽  
R. Ziegler ◽  
J. Schaub
Keyword(s):  
Column Chromatography ◽  
Deae Cellulose ◽  
Deae Cellulose Column ◽  
Cellulose Column

In vitro 19-norandrogen synthesis by equine placenta requires the participation of aromatase

1995 ◽  
Vol 144 (3) ◽  
pp. 517-525 ◽  
Author(s):  
S Moslemi ◽  
P Silberzahn ◽  
J-L Gaillard
Keyword(s):  
Silica Gel ◽  
Aromatase Inhibitors ◽  
Column Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Reverse Phase Hplc ◽  
Reverse Phase ◽  
Term Placenta ◽  
Flow Detector

Abstract Explants of equine full-term placenta have been shown to synthesize 19-norandrogens from labelled androgens. Steroid metabolites were purified by silica-gel column chromatography then analysed and quantified by C18-reverse-phase HPLC coupled to a radioactive flow detector. 19-Norandrostenedione was subsequently recrystallized to constant specific activity, providing unequivocal evidence of its synthesis by the equine placenta. 19-Norandrostenedione synthesis appeared to be localized in the microsomal fraction. Regardless of the substrate used, formation of 19-norandrogens was far weaker than that of oestrogens; moreover, the yield of 17-oxosteroids produced was much greater than that of 17β-hydroxysteroids, suggesting the presence of a dehydrogenase with predominant oxidative activity. Sulphoconjugated steroids formed were less than 0·5% of total steroids. Although 19-nortestosterone could not be generated by equine purified aromatase incubated with labelled testosterone, the synthesis of 19-norandrogens and oestrogens by equine placental explants was blocked by two specific aromatase inhibitors, 4-hydroxyandrostenedione and fadrozole. Our results provide evidence for a placental origin of at least a part of the 19-norandrogens previously identified in the blood of the pregnant mare. Furthermore, it is suggested that 19-norandrogen biosynthesis would involve the enzymatic metabolism of 19-oxygenated androgens formed by equine aromatase. Journal of Endocrinology (1995) 144, 517–525

Vimentin Intermediate Filament Formation: In Vitro Measurement and Mathematical Modeling of the Filament Length Distribution during Assembly†

Langmuir ◽  
2009 ◽  
Vol 25 (15) ◽  
pp. 8817-8823 ◽  
Author(s):  
Stéphanie Portet ◽  
Norbert Mücke ◽  
Robert Kirmse ◽  
Jörg Langowski ◽  
Michael Beil ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Intermediate Filament ◽  
Length Distribution ◽  
Filament Length ◽  
Filament Formation ◽  
Vimentin Intermediate Filament

Upgrading wheat straw with urea at a tropical temperature: effects of amount of urea and moisture on in vitro digestibility and pH

1996 ◽  
Vol 1996 ◽  
pp. 212-212
Author(s):  
I.U. Haq ◽  
E. Owen
Keyword(s):  
Wheat Straw ◽  
Urea Solution ◽  
In Vitro Digestibility ◽  
Ammonia Treatment ◽  
Urea Treatment ◽  
Cow Urine ◽  
The Cost ◽  
The Tropics ◽  
On Farm

Urea-ammonia treatment of straws in the tropics involves mixing 1.0 kg of air dry straw with 1.0 kg of a 40 g/kg urea solution and storing under plastic for at least 4 weeks (Schiere and Ibrahim, 1989). The economics of treatment is dependent on the cost of urea. Treatment cost would reduce, if on-farm-produced urine, e.g. cow urine, could be used as a source of urea. However cow urine is dilute and may contain only 10 g/kg urea or less (Owen, 1993). The present study therefore investigated varying concentrations of urea solution for treating wheat straw at a tropical temperature.

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