scholarly journals On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone

1983 ◽  
Vol 215 (1) ◽  
pp. 191-199 ◽  
Author(s):  
S Synouri-Vrettakou ◽  
K A Mitropoulos
Keyword(s):  
Microsomal Fraction ◽  
Rate Of Change ◽  
Time Dependent ◽  
Cholesterol Concentration ◽  
Phosphatidylcholine Liposomes ◽  
Acat Activity ◽  
Rate Of Increase ◽  
A Site

The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.

scholarly journals Conditions that may result in (de-)phosphorylation of hepatic acyl-CoA:cholesterol acyltransferase result also in modulation of substrate supply in vitro

1984 ◽  
Vol 221 (3) ◽  
pp. 685-695 ◽  
Author(s):  
K A Mitropoulos ◽  
S Venkatesan
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Covalent Modification ◽  
Time Dependent ◽  
Cholesterol Concentration ◽  
Acat Activity ◽  
Phospholipid Liposomes ◽  
Substrate Supply ◽  
Rate Of Increase ◽  
Liver Microsomal Fraction

The present experiments were designed to study intervesicular transfer of cholesterol in rat liver microsomal fraction and modulation of the activity of acyl-CoA:cholesterol acyltransferase (ACAT) under conditions that are expected to result in the covalent modification (phosphorylation/dephosphorylation) of the enzyme. Preincubation of rat liver microsomal fraction followed by assay of ACAT showed a time-dependent increase in activity. This rate was temperature-dependent. Preincubation in the presence of cholesterol/phospholipid liposomes resulted in a time-dependent transfer of cholesterol from liposomal to the microsomal vesicles and in an increase in the rate of ACAT change owing to the preincubation. Both these rates were dependent on liposomal cholesterol concentration and on temperature. The presence of cytosol in the preincubation mixture increased the rate of change of ACAT activity in the absence or in the presence of cholesterol/phospholipid liposomes. In the latter case the presence of cytosol also increased the rate of transfer of cholesterol from liposomal to the microsomal vesicles. Activation energies of the rate of this transfer and of the rate of increase of ACAT activity were similar in the presence and in the absence of cytosol. Both in the absence and in the presence of cytosol, the presence of NaF (50 mM) in the preincubation mixture considerably decreased the rate of transfer of cholesterol from liposomal to microsomal vesicles and the rate of increase of ACAT activity. The presence of Mg2+ in the preincubation mixture produced no effect on the rate of transfer of cholesterol from liposomal to the microsomal vesicles, although under most conditions it decreased the rate of increase of ACAT activity caused by the preincubation. These results are discussed in relation to the molecular mechanism involved in this intervesicular transfer of cholesterol and to the modulation of ACAT activity by substrate supply, and also in relation to the hypothesis that ACAT activity can be modulated by a mechanism involving the phosphorylation/dephosphorylation of the enzyme.

scholarly journals Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro

1980 ◽  
Vol 186 (2) ◽  
pp. 591-598 ◽  
Author(s):  
Christopher Kirby ◽  
Jacqui Clarke ◽  
Gregory Gregoriadis
Keyword(s):  
Blood Plasma ◽  
Intravenous Injection ◽  
Whole Blood ◽  
Cholesterol Content ◽  
Molar Ratio ◽  
Phosphatidylcholine Liposomes ◽  
Effective Use ◽  
Positively Charged

Small unilamellar neutral, negatively and positively charged liposomes composed of egg phosphatidylcholine, various amounts of cholesterol and, when appropriate, phosphatidic acid or stearylamine and containing 6-carboxyfluorescein were injected into mice, incubated with mouse whole blood, plasma or serum or stored at 4°C. Liposomal stability, i.e. the extent to which 6-carboxyfluorescein is retained by liposomes, was dependent on their cholesterol content. (1) Cholesterol-rich (egg phosphatidylcholine/cholesterol, 7:7 molar ratio) liposomes, regardless of surface charge, remained stable in the blood of intravenously injected animals for up to at least 400min. In addition, stability of cholesterol-rich liposomes was largely maintained in vitro in the presence of whole blood, plasma or serum for at least 90min. (2) Cholesterol-poor (egg phosphatidylcholine/cholesterol, 7:2 molar ratio) or cholesterol-free (egg phosphatidylcholine) liposomes lost very rapidly (at most within 2min) much of their stability after intravenous injection or upon contact with whole blood, plasma or serum. Whole blood and to some extent plasma were less detrimental to stability than was serum. (3) After intraperitoneal injection, neutral cholesterol-rich liposomes survived in the peritoneal cavity to enter the blood circulation in their intact form. Liposomes injected intramuscularly also entered the circulation, although with somewhat diminished stability. (4) Stability of neutral and negatively charged cholesterol-rich liposomes stored at 4°C was maintained for several days, and by 53 days it had declined only moderately. Stored liposomes retained their unilamellar structure and their ability to remain stable in the blood after intravenous injection. (5) Control of liposomal stability by adjusting their cholesterol content may help in the design of liposomes for effective use in biological systems in vivo and in vitro.

scholarly journals A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

1991 ◽  
Vol 114 (4) ◽  
pp. 773-786 ◽  
Author(s):  
P D Kouklis ◽  
T Papamarcaki ◽  
A Merdes ◽  
S D Georgatos
Keyword(s):  
Potential Role ◽  
Type Iii ◽  
Filament Assembly ◽  
Filament Bundles ◽  
Self Association ◽  
Specific Association ◽  
A Site ◽  
Idiotypic Antibody

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

scholarly journals In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

1990 ◽  
Vol 10 (8) ◽  
pp. 3868-3872 ◽  
Author(s):  
C M Shumard ◽  
C Torres ◽  
D C Eichler
Keyword(s):  
18S Rrna ◽  
Precise Determination ◽  
In Vivo Studies ◽  
Rrna Sequence ◽  
S1 Nuclease ◽  
In Vitro Processing ◽  
Rrna Maturation ◽  
A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

scholarly journals Metformin Inhibited Growth, Invasion and Metastasis of Esophageal Squamous Cell Carcinoma in Vitro and in Vivo

2018 ◽  
Vol 51 (3) ◽  
pp. 1276-1286 ◽  
Author(s):  
Feng Liang ◽  
Yu-Gang Wang ◽  
Changcheng Wang
Keyword(s):  
Squamous Cell Carcinoma ◽  
Plasma Glucose Level ◽  
Caspase 3 ◽  
Time Dependent ◽  
Dependent Manner ◽  
Metformin Treatment ◽  
Invasion And Migration ◽  
And Migration

Background/Aims: This study aimed at investigating the effects of metformin on the growth and metastasis of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo. Methods: Two human ESCC cell lines EC9706 and Eca109 were selected and challenged with metformin in this study. Western blot assay was performed to detect th level of Bcl-2, Bax and Caspase-3. Scratch wound assay, transwell assay and Millicell invasion assay were used to assay the invasion and migration of EC9706 and Eca109 cells. Nude mice tumor models were used to assay the growth and lung metastasis of ESCC cells after metformin treatment. The plasma glucose level was also assayed. Results: We found that metformin significantly inhibited proliferation and induced apoptosis of both ESCC cell lines in a dose- and time-dependent manner, and the expression of Bcl-2 was down-regulated and Bax and Caspase-3 were up-regulated. Metformin significantly inhibited the invasion and migration of EC9706 and Eca109 cells (p < 0.05). mRNA and protein levels of MMP-2 and MMP-9 decreased significantly upon treatment with metformin of 10mM for 12, 24 and 48h in a time-dependent manner (p < 0.05). In line with in vitro results, in vivo experiments demonstrated that metformin inhibited tumorigenicity, inhibited lung metastasis and down-regulated the expression of MMP-2 and MMP-9. Moreover, we showed that metformin treatment did not cause significant alteration in liver and renal functions and plasma glucose level. Conclusion: Our study for the first time demonstrated the anti-invasive and anti-metastatic effects of metformin on human ESCC cells both in vitro and in vivo, which might be associated with the down-regulation of MMP-2 and MMP-9. As a whole, our results indicate the potential of metformin to be developed as a chemotherapeutic agent for patients with ESCC and might stimulate future studies on this area.

Preparative enrichment of human tissue cells capable to change a site of growth in vitro or in vivo - Recent developments

2018 ◽  
Vol 48 (10) ◽  
pp. 954-960 ◽  
Author(s):  
Johann Bauer ◽  
Hari H. P. Cohly ◽  
Jayashree Sahana ◽  
Daniela Grimm
Keyword(s):  
Human Tissue ◽  
Recent Developments ◽  
Tissue Cells ◽  
A Site

scholarly journals The Escherichia coli cysG promoter belongs to the ‘extended −10’ class of bacterial promoters

1993 ◽  
Vol 296 (3) ◽  
pp. 851-857 ◽  
Author(s):  
T Belyaeva ◽  
L Griffiths ◽  
S Minchin ◽  
J Cole ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Growth Conditions ◽  
Upstream Region ◽  
E Coli ◽  
Run Off ◽  
A Site ◽  
Specific Sequences

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5′-TGN-3′ motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the ‘extended -10’ class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

scholarly journals 3'-end-forming signals of yeast mRNA.

1995 ◽  
Vol 15 (11) ◽  
pp. 5983-5990 ◽  
Author(s):  
Z Guo ◽  
F Sherman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cleavage Sites ◽  
Yeast Saccharomyces Cerevisiae ◽  
Higher Eukaryotes ◽  
A Site

It was previously shown that three distinct but interdependent elements are required for 3' end formation of mRNA in the yeast Saccharomyces cerevisiae: (i) the efficiency element TATATA and related sequences, which function by enhancing the efficiency of positioning elements; (ii) positioning elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation. In this study, we have shown that several A-rich sequences, including the vertebrate poly(A) signal AATAAA, are also positioning elements. Saturated mutagenesis revealed that optimum sequences of the positioning element were AATAAA and AAAAAA and that this element can tolerate various extents of replacements. However, the GATAAA sequence was completely ineffective. The major cleavage sites determined in vitro corresponded to the major poly(A) sites observed in vivo. Our findings support the assumption that some components of the basic polyadenylation machinery could have been conserved among yeasts, plants, and mammals, although 3' end formation in yeasts is clearly distinct from that of higher eukaryotes.

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