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Fertility and Sterility Mar 1985Autoantibodies to thyroid microsomes were more frequent in 102 infertile men with complement-dependent sperm-immobilizing activity (sperm immobilization test [SIT] ) in...
Autoantibodies to thyroid microsomes were more frequent in 102 infertile men with complement-dependent sperm-immobilizing activity (sperm immobilization test [SIT] ) in serum (11.8%) than in a control group of 277 men of comparable ages and semen quality without sperm antibodies (4.3%, P less than 0.05). Frequencies of organ-specific antibodies (antigastric parietal cell, antithyroglobulin, and antithyroid microsome) in 57 men with genital tract obstructions and positive SIT were similar to those for control subjects, and there were no significant differences in the frequencies of non-organ-specific autoantibodies (antinuclear antibody, rheumatoid factor, antimitochondrial, and anti-smooth muscle) in the three groups. Because in men without genital tract obstruction antithyroid microsomal autoantibodies were more common with sperm antibodies than without, the possibility of a genetic factor in the causation of sperm autoimmunity should be considered.
Topics: Adult; Antibodies; Autoantibodies; Humans; Infertility, Male; Male; Microsomes; Middle Aged; Sperm Motility; Spermatozoa; Thyroglobulin; Thyroid Gland
PubMed: 3979583
DOI: 10.1016/s0015-0282(16)48445-1 -
Biochimica Et Biophysica Acta May 2014Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic...
Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.
Topics: Animals; Biomimetic Materials; Biomimetics; Cell-Free System; Insecta; Lipids; Liposomes; Membrane Proteins; Membranes; Microsomes; Models, Biological; Unilamellar Liposomes
PubMed: 24370776
DOI: 10.1016/j.bbamem.2013.12.009 -
The Biochemical Journal Nov 19651. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the...
1. The activities of microsome fractions from the liver of adult and 5-day-old rats for the incorporation of [(14)C]phenylalanine into protein were similar in the presence and absence of polyuridylic acid. 2. The activity of a light-microsome fraction from adult liver was greater than that of a heavy-microsome fraction, and the light-microsome fraction was also more markedly stimulated by the presence of polyuridylic acid. 3. The light-microsome fraction, when analysed by density-gradient centrifugation, contained a higher ratio of free ribosomes to bound ribosomes, whereas the reverse was true for the heavy-microsome fraction. Similar results were obtained for liver from adult and 5-day-old rats. 4. When the light-microsome fraction was incubated under conditions in which amino acid was incorporated into protein there was only a small increase in the ratio of free to bound ribosomes. When such a fraction was incubated with [(14)C]leucine and was then subjected to density-gradient centrifugation the fraction with the highest specific activity based on RNA had a density between that of the bound and free ribosomes. Treatment of the incubated fraction with ribonuclease shifted the radioactivity towards the free ribosome peak. These properties are consistent with the presence of active free polysomes. Such a component appeared also to be present when the heavy-microsome fraction was incubated under similar conditions. 5. The effect of the presence of polyuridylic acid on the incorporation of [(14)C]phenylalanine by the light-microsome fractions from liver of adult and 5-day-old rats was greatest in the region of the free ribosomes, but it is probable that some small polysomes containing polyuridylic acid are formed. 6. Polyuridylic acid also stimulated the bound ribosomes to a small extent when the heavy-microsome fraction from the liver of young rats was incubated with [(14)C]phenylalanine. 7. The results are discussed in terms of the various morphological constituents in liver now known to play a role in the synthesis of protein for export and for the internal activity of the cell.
Topics: Animals; In Vitro Techniques; Liver; Microscopy, Electron; Microsomes; Phenylalanine; Polynucleotides; Protein Biosynthesis; Rats; Ribosomes; Subcellular Fractions; Uracil Nucleotides
PubMed: 5880015
DOI: 10.1042/bj0970422 -
Drug Metabolism and Disposition: the... Jul 2019Nonspecific drug partitioning into microsomal membranes must be considered for in vitro-in vivo correlations. This work evaluated the effect of including lipid...
Nonspecific drug partitioning into microsomal membranes must be considered for in vitro-in vivo correlations. This work evaluated the effect of including lipid partitioning in the analysis of complex TDI kinetics with numerical methods. The covariance between lipid partitioning and multiple inhibitor binding was evaluated. Simulations were performed to test the impact of lipid partitioning on the interpretation of TDI kinetics, and experimental TDI datasets for paroxetine (PAR) and itraconazole (ITZ) were modeled. For most kinetic schemes, modeling lipid partitioning results in statistically better fits. For MM-IL simulations (K = 0.1 M, k = 0.1 minute), concurrent modeling of lipid partitioning for an f range (0.01, 0.1, and 0.5) resulted in better fits compared with post hoc correction (AICc: -526 vs. -496, -579 vs. -499, and -636 vs. -579, respectively). Similar results were obtained with EII-IL. Lipid partitioning may be misinterpreted as double binding, leading to incorrect parameter estimates. For the MM-IL datasets, when f = 0.02, MM-IL, and EII model fits were indistinguishable (AICc = 3). For less partitioned datasets (f = 0.1 or 0.5), the inclusion of partitioning resulted in better models. The inclusion of lipid partitioning can lead to markedly different estimates of K and k A reasonable alternate experimental design is nondilution TDI assays, with post hoc f incorporation. The best fit models for PAR (MIC-M-IL) and ITZ (MIC-EII-M-IL and MIC-EII-M-Seq-IL) were consistent with their reported mechanism and kinetics. Overall, experimental f values should be concurrently incorporated into TDI models with complex kinetics, when dilution protocols are used.
Topics: Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Humans; Itraconazole; Lipid Metabolism; Microsomes; Models, Biological; Paroxetine
PubMed: 31043439
DOI: 10.1124/dmd.118.085969 -
Drug Metabolism and Disposition: the... Oct 20164-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and...
4-Ipomeanol (IPO) is a model pulmonary toxicant that undergoes P450-mediated metabolism to reactive electrophilic intermediates that bind to tissue macromolecules and can be trapped in vitro as the NAC/NAL adduct. Pronounced species and tissue differences in IPO toxicity are well documented, as is the enzymological component of phase I bioactivation. However, IPO also undergoes phase II glucuronidation, which may compete with bioactivation in target tissues. To better understand the organ toxicity of IPO, we synthesized IPO-glucuronide and developed a new quantitative mass spectrometry-based assay for IPO glucuronidation. Microsomal rates of glucuronidation and P450-dependent NAC/NAL adduct formation were compared in lung, kidney, and liver microsomes from seven species with different target organ toxicities to IPO. Bioactivation rates were highest in pulmonary and renal microsomes from all animal species (except dog) known to be highly susceptible to the extrahepatic toxicities induced by IPO. In a complementary fashion, pulmonary and renal IPO glucuronidation rates were uniformly low in all experimental animals and primates, but hepatic glucuronidation rates were high, as expected. Therefore, with the exception of the dog, the balance between microsomal NAC/NAL adduct and glucuronide formation correlate well with the risk for IPO-induced pulmonary, renal, and hepatic toxicities across species.
Topics: Animals; Cattle; Dogs; Female; Glucuronides; Humans; Kidney; Liver; Lung; Macaca fascicularis; Male; Mice, Inbred C57BL; Microsomes; Oxidation-Reduction; Rabbits; Rats; Rats, Sprague-Dawley; Species Specificity; Terpenes
PubMed: 27468999
DOI: 10.1124/dmd.116.070003 -
Scientific Reports Sep 2017Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called "difficult-to-express" proteins...
Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called "difficult-to-express" proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.
Topics: Animals; CHO Cells; Cell-Free System; Cricetulus; Membrane Proteins; Microsomes; Proteins; Single-Chain Antibodies
PubMed: 28916746
DOI: 10.1038/s41598-017-12188-8 -
The Biochemical Journal Jan 19691. A study was made of the hydroxylation of trans-stilbene in rabbits, guinea pigs and mice, as well as by rabbit liver microsomes. 2. In the rabbit in vivo,...
1. A study was made of the hydroxylation of trans-stilbene in rabbits, guinea pigs and mice, as well as by rabbit liver microsomes. 2. In the rabbit in vivo, trans-stilbene is converted into 4-hydroxy-,4,4'-dihydroxy-,3-hydroxy-4-methoxy-and 4-hydroxy-3-methoxy-stilbene, and hydroxylation plays a more significant role in the metabolism of trans-stilbene than has previously been reported. 3. Investigation of the hydroxylation of 4-hydroxystilbene in the rabbit in vivo demonstrated its ready conversion into 4,4'-dihydroxystilbene and established its intermediacy in the formation of this compound and the methylated analogues of 3,4-dihydroxystilbene. 4. Hydroxylation of trans-stilbene in the guinea pig was found to follow a pattern similar, both qualitatively and quantitatively, to that in the rabbit. 5. Studies in the mouse revealed only limited yields of 4,4'-dihydroxystilbene. 6. Studies of the hydroxylation of trans-stilbene and 4-hydroxystilbene by rabbit liver microsomes located two of the reactions that occur with these compounds in vivo. 7. Work with a solubilized liver-microsomal preparation provided evidence that ;stilbene hydroxylase' activity is not completely lost on solubilization, thus allowing for future microsomal enzyme-isolation studies.
Topics: Animals; Chromatography, Paper; Chromatography, Thin Layer; Guinea Pigs; In Vitro Techniques; Liver; Mice; Microsomes; Mixed Function Oxygenases; Rabbits; Stilbenes
PubMed: 5775688
DOI: 10.1042/bj1110035 -
FEBS Letters May 1994N-Oligosaccharyltransferase catalyzes the N-glycosylation of asparagine residues of nascent polypeptide chains in the endoplasmic reticulum, a pathway highly conserved... (Comparative Study)
Comparative Study
N-Oligosaccharyltransferase catalyzes the N-glycosylation of asparagine residues of nascent polypeptide chains in the endoplasmic reticulum, a pathway highly conserved in all eukaryotes. An enzymatically active complex was isolated from microsomal membranes from Saccharomyces cerevisiae, which is composed of four proteins: Wbp1p and Swp1p (previously found to be encoded by two essential genes necessary for N-glycosylation in vivo and in vitro) and two additional proteins with a molecular mass of 60/62 kDa and 34 kDa. The 60/62 component represents differentially glycosylated forms of a protein that has sequence homology to ribophorin I. Wbp1p and Swp1p reveal homology to mammalian OST 48 and ribophorin II, respectively. Ribophorin I and II and OST 48 were recently shown to be constituents of the mammalian transferase from dog pancreas. The data reveal a high conservation of the organization of this enzyme activity.
Topics: Amino Acid Sequence; Animals; Asparagine; Dogs; Glycosylation; Hexosyltransferases; Intracellular Membranes; Membrane Proteins; Microsomes; Molecular Sequence Data; Pancreas; Saccharomyces cerevisiae; Sequence Homology; Transferases
PubMed: 8181570
DOI: 10.1016/0014-5793(94)00356-4 -
BMC Pharmacology Oct 2004Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an...
BACKGROUND
Endogenous nitric oxide (NO) and carbon monoxide (CO) are generated by nitric oxide synthase and heme oxygenase, respectively. Like NO, CO has been accepted as an important cellular signaling molecule in biological systems. An up-regulation in both gene and protein expression of heme oxygenase-1 (HO-1) under oxidative/nitrosative stress has been well documented, and the protective role of HO-1 and HO-2 against oxidative damage is proposed. However, data on the direct effect of reactive oxygen/nitrogen species (ROS/RNS) on HO function is incomplete. Using gas chromatography to quantify carbon monoxide (CO) formation from heme oxidation, we investigated the effects of peroxynitrite (ONOO-) on the in vitro catalytic activity of rat spleen (HO-1) and brain (HO-2) microsomal heme oxygenases.
RESULTS
Exposure to ONOO- led to concentration-dependent but reversible decreases in the activity of microsomal rat spleen and brain HO activity. Spleen HO activity was 100-fold more sensitive to ONOO--dependent inactivation compared to that of the brain, with IC50 values of 0.015 +/- 0.005 mM and 1.25 +/- 0.25 mM respectively. Inhibition of both rat spleen and brain microsomal HO activity was also observed with tetra-nitromethane, a tyrosine nitrating agent, as well as two NO donors, S-nitrosoglutathione (GSNO) and diethylamine NONOate (DEA-NONOate). However, no additive effect was found following the application of NO donors and ONOO- together.
CONCLUSION
These results indicate that ONOO- may regulate HO-1 and HO-2 activities by mechanisms that involve different interactions with these proteins. It is suggested that while nitration of tyrosine residues and oxidation of sulfhydryl groups may be involved, consideration should be given to other facets of ONOO- chemistry. This inhibition of HO activity offers a mechanism for cross talk between the nitric oxide synthase and HO systems.
Topics: Animals; Carbon Monoxide; Dose-Response Relationship, Drug; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; In Vitro Techniques; Male; Microsomes; Nitric Oxide; Nitrites; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Sulfhydryl Compounds
PubMed: 15498099
DOI: 10.1186/1471-2210-4-26 -
Anesthesiology Aug 2001Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation....
BACKGROUND
Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE). The free radical is also thought to initiate lipid peroxidation. Halothane-dependent lipid peroxidation has been shown in animals in vitro and in vivo but has not been evaluated in humans. This investigation tested the hypothesis that halothane causes lipid peroxidation in human liver microsomes, identified P450 isoforms responsible for halothane-dependent lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction.
METHODS
Halothane metabolism was determined using human liver microsomes or cDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyde (MDA) formation using high-pressure liquid chromatography-ultraviolet analysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry.
RESULTS
Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA production exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production was catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone. CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2A6 antibody.
CONCLUSIONS
Halothane causes lipid peroxidation in human liver microsomes, which is catalyzed by CYP2A6, and inhibition of halothane reduction prevents halothane-dependent lipid peroxidation in vitro.
Topics: Anesthetics, Inhalation; Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2A6; Cytochrome P-450 Enzyme System; Halothane; Humans; In Vitro Techniques; Isoenzymes; Lipid Peroxidation; Malondialdehyde; Microsomes, Liver; Mixed Function Oxygenases; Oxidation-Reduction; Rats
PubMed: 11506127
DOI: 10.1097/00000542-200108000-00037