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Journal of Enzyme Inhibition and... Dec 2022Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability.... (Comparative Study)
Comparative Study
Esters are one of the major functional groups present in the structures of prodrugs and bioactive compounds. Their presence is often associated with hydrolytic lability. In this paper, we describe a comparative chemical and biological stability of homologous esters and isosteres in base media as well as in rat plasma and rat liver microsomes. Our results provided evidence for the hydrolytic structure lability relationship and demonstrated that the hydrolytic stability in plasma and liver microsome might depend on carboxylesterase activity. Molecular modelling studies were performed in order to understand the experimental data. Taken together, the data could be useful to design bioactive compounds or prodrugs based on the correct choice of the ester subunit, addressing compounds with higher or lower metabolic lability.
Topics: Animals; Carboxylesterase; Dose-Response Relationship, Drug; Enzyme Inhibitors; Esters; Hydrolysis; Male; Microsomes, Liver; Models, Molecular; Molecular Structure; Prodrugs; Rats; Rats, Wistar; Structure-Activity Relationship
PubMed: 35156494
DOI: 10.1080/14756366.2022.2027933 -
ACS Chemical Biology Dec 2019The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this...
The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this study, the possibility of enhancing a whole cell based antibacterial library screen by increasing the dimensionality of the screening effort is explored using methicillin-resistant (MRSA) as the target organism. One dimension involved generating and screening a human liver microsome metabolized FDA approved drug library. Comparative screening of the un-metabolized (UM) and pre-metabolized (PM) libraries allows identification of intrinsically active agents from the UM library screen and of agents with active metabolites from the PM library screen. To further enhance this screening effort, it was combined with a -/+ resistant to antibiotic screen (-/+ cefoxitin; Cef). This allows the identification of agents that can act synergistically with the resistant to antibiotic. This approach revealed five compounds with substantially improved activity after metabolism and four compounds with substantial synergistic activity with cefoxitin. Capecitabine in particular only had significant antibacterial activity after metabolism. Its metabolites were isolated, identified, and characterized for spectrum of activity along with several other anticancer drugs with anti-MRSA activity. Floxuridine, gemcitabine, novobiocin, and rifaximin were identified as having substantial synergy with cefoxitin from the -/+Cef screens. Checkerboard assays verified synergy for these agents. Floxuridine demonstrated a particularly high degree of synergy with cefoxitin (FIC = 0.14). This study demonstrates how a dimensionally enhanced comparative screening effort can identify new antibacterial agents and strategies for countering antibacterial agent resistance.
Topics: Anti-Bacterial Agents; Humans; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Microsomes, Liver
PubMed: 31675203
DOI: 10.1021/acschembio.9b00745 -
Molecules (Basel, Switzerland) Dec 2020Several natural-based compounds and products are reported to possess anti-inflammatory and immunomodulatory activity both in vitro and in vivo. The primary target for... (Review)
Review
Present Status and Future Trends of Natural-Derived Compounds Targeting T Helper (Th) 17 and Microsomal Prostaglandin E Synthase-1 (mPGES-1) as Alternative Therapies for Autoimmune and Inflammatory-Based Diseases.
Several natural-based compounds and products are reported to possess anti-inflammatory and immunomodulatory activity both in vitro and in vivo. The primary target for these activities is the inhibition of eicosanoid-generating enzymes, including phospholipase A2, cyclooxygenases (COXs), and lipoxygenases, leading to reduced prostanoids and leukotrienes. Other mechanisms include modulation of protein kinases and activation of transcriptases. However, only a limited number of studies and reviews highlight the potential modulation of the coupling enzymatic pathway COX-2/mPGES-1 and Th17/Treg circulating cells. Here, we provide a brief overview of natural products/compounds, currently included in the Italian list of botanicals and the BELFRIT, in different fields of interest such as inflammation and immunity. In this context, we focus our opinion on novel therapeutic targets such as COX-2/mPGES-1 coupling enzymes and Th17/Treg circulating repertoire. This paper is dedicated to the scientific career of Professor Nicola Mascolo for his profound dedication to the study of natural compounds.
Topics: Anti-Inflammatory Agents; Autoimmune Diseases; Biological Products; Complementary Therapies; Cyclooxygenase 1; Cyclooxygenase 2; Humans; Inflammation; Microsomes; Th17 Cells
PubMed: 33353211
DOI: 10.3390/molecules25246016 -
Drug Metabolism and Disposition: the... May 2017In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant...
In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of subcellular fractions rely on robust estimates of physiologically relevant scaling factors for the prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery marker and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase (G6Pase) activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g in dog kidney cortex, and 41.1 and 63.6 mg/g in dog liver ( = 17), using P450 content and G6Pase activity, respectively. No trends were noted between kidney, liver, and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g in kidney cortex ( = 38), respectively. MPPGK was 2-fold greater than the commonly used in vitro-in vivo extrapolation scalar; this difference was attributed mainly to tissue source (mixed kidney regions versus cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex versus whole kidney-specific MPPGK, tissue weight, and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo.
Topics: Animals; Cytochrome P-450 Enzyme System; Cytosol; Dogs; Female; Glucose-6-Phosphatase; Humans; Kidney Cortex; Male; Microsomes; Species Specificity
PubMed: 28270564
DOI: 10.1124/dmd.117.075242 -
Journal of Lipid Research Oct 2008Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids.... (Review)
Review
Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids. The existence of multiple enzyme isoforms with GPAT activity was predicted many years ago when GPAT activities with distinct kinetic profiles and sensitivity to inhibitors were characterized in two subcellular compartments, mitochondria and microsomes. We now know that mammals have at least four GPAT isoforms with distinct tissue distribution and function. GPAT1 is the major mitochondrial GPAT isoform and is characterized by its resistance to sulfhydryl-modifying reagents, such as N-ethylmaleimide (NEM). GPAT2 is a minor NEM-sensitive mitochondrial isoform. The activity referred to as microsomal GPAT is encoded by two closely related genes, GPAT3 and GPAT4. GPAT isoforms are important regulators of cellular triglyceride and phospholipid content, and may channel fatty acids toward particular metabolic fates. Overexpression and knock-out studies suggest that GPAT isoforms can play important roles in the development of hepatic steatosis, insulin resistance, and obesity; GPAT isoforms are also important for lactation. This review summarizes the current state of knowledge on mammalian GPAT isoforms.
Topics: Amino Acid Motifs; Amino Acid Sequence; Animals; Ethylmaleimide; Glycerol-3-Phosphate O-Acyltransferase; Humans; Isoenzymes; Microsomes
PubMed: 18658143
DOI: 10.1194/jlr.R800013-JLR200 -
BMC Plant Biology Jan 2021Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain...
BACKGROUND
Simmondsia chinensis (jojoba) is the only plant known to store wax esters instead of triacylglycerols in its seeds. Wax esters are composed of very-long-chain monounsaturated fatty acids and fatty alcohols and constitute up to 60% of the jojoba seed weight. During jojoba germination, the first step of wax ester mobilization is catalyzed by lipases. To date, none of the jojoba lipase-encoding genes have been cloned and characterized. In this study, we monitored mobilization of storage reserves during germination of jojoba seeds and performed detailed characterization of the jojoba lipases using microsomal fractions isolated from germinating seeds.
RESULTS
During 26 days of germination, we observed a 60-70% decrease in wax ester content in the seeds, which was accompanied by the reduction of oleosin amounts and increase in glucose content. The activity of jojoba lipases in the seed microsomal fractions increased in the first 50 days of germination. The enzymes showed higher activity towards triacylglycerols than towards wax esters. The maximum lipase activity was observed at 60 °C and pH around 7 for triacylglycerols and 6.5-8 for wax esters. The enzyme efficiently hydrolyzed various wax esters containing saturated and unsaturated acyl and alcohol moieties. We also demonstrated that jojoba lipases possess wax ester-synthesizing activity when free fatty alcohols and different acyl donors, including triacylglycerols and free fatty acids, are used as substrates. For esterification reactions, the enzyme utilized both saturated and unsaturated fatty alcohols, with the preference towards long chain and very long chain compounds.
CONCLUSIONS
In in vitro assays, jojoba lipases catalyzed hydrolysis of triacylglycerols and different wax esters in a broad range of temperatures. In addition, the enzymes had the ability to synthesize wax esters in the backward reaction. Our data suggest that jojoba lipases may be more similar to other plant lipases than previously assumed.
Topics: Caryophyllales; Esters; Germination; Hydrolysis; Lipase; Lipids; Microsomes; Orlistat; Plant Proteins; Seeds; Substrate Specificity; Temperature; Triglycerides; Waxes
PubMed: 33468064
DOI: 10.1186/s12870-020-02823-4 -
The Journal of Biophysical and... Mar 1956Rat liver, liver homogenates, and microsome fractions separated therefrom were examined systematically in the electron microscope in sections of OsO(4)-fixed,...
Rat liver, liver homogenates, and microsome fractions separated therefrom were examined systematically in the electron microscope in sections of OsO(4)-fixed, methacrylate-embedded tissue and pellets. It was found that most microsomes are morphologically identical with the rough surfaced elements of the endoplasmic reticula of hepatic cells. They appear as isolated, membrane-bound vesicles, tubules, and cisternae which contain an apparently homogeneous material of noticeable density, and bear small, dense particles (100 to 150 A) attached to their outer aspect. In solutions of various osmolar concentrations they behave like osmometers. The findings suggest that they derive from the endoplasmic reticulum by a generalized pinching-off process rather than by mechanical fragmentation. The microsome fractions contain in addition relatively few vesicles free of attached particles, probably derived from the smooth surfaced parts of the endoplasmic reticula. Dense, peribiliary bodies represent a minor component of the same fractions. The microsomes derived from 1 gm. wet weight liver pulp contained (averages of 10 experiments) 3.09 mg. protein N, 3.46 mg. RNA (RNA/protein N = 1.12), and 487 microg. phospholipide P. They displayed DPNH-cytochrome c reductase activity and contained an alcohol-soluble hemochromogen. The microsome preparations proved resistant to washing and "aging." Treatment with versene and incubation with ribonuclease (30 minutes at 37 degrees C.) resulted in appreciable losses of RNA and in partial or total disappearance of attached particles. Treatment with deoxycholate (0.3 to 0.5 per cent, pH = 7.5) induced a partial clarification of the microsome suspensions which, upon centrifugation, yielded a small pellet of conglomerated small, dense particles (100 to 150 A) with only occasionally interspersed vesicles. The pellet contained approximately 80 to 90 per cent of the RNA and approximately 20 per cent of the protein N of the original microsomes. The supernatant accounted satisfactorily for the materials lost during deoxycholate treatment. The findings suggest that the microsomal RNA is associated with the small particles whereas most of the protein and nearly all of the phospholipide, hemochromogen, and DPNH-cytochrome c reductase activity are associated with the membrane or content of the microsomes.
Topics: Animals; Centrifugation; Endoplasmic Reticulum; Liver; Membranes; Microsomes, Liver; Phospholipids; Proteins; RNA; Rats
PubMed: 13319380
DOI: 10.1083/jcb.2.2.171 -
The Journal of Cell Biology Jan 1962Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between...
Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 microg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry.
Topics: Animals; Membranes; Microsomes; Microsomes, Liver; Nucleoproteins; Phospholipids; RNA; Rats; Ribosomes
PubMed: 13878497
DOI: 10.1083/jcb.12.1.17 -
TheScientificWorldJournal 2016The present study aimed to investigate the mutagenic activity of essential oil. The most abundant compounds identified by GC-MS were -terpinene (25.73%), -terpinene...
The present study aimed to investigate the mutagenic activity of essential oil. The most abundant compounds identified by GC-MS were -terpinene (25.73%), -terpinene (17.35%), terpinen-4-ol (17.24%), and sabinene (10.8%). Mutagenicity was evaluated by the /microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535 strains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 L/plate in the absence of S9 mix and higher than 0.08 L/plate in the presence of S9 mix and no gene mutation increase was observed. For the mammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 g/mL. Moreover, when tested in noncytotoxic concentrations, essential oil was not able to induce chromosome mutation. The results from this study therefore suggest that essential oil is not mutagenic at the concentrations tested in the /microsome and micronucleus assays.
Topics: Animals; Cells, Cultured; Cricetinae; Fibroblasts; Micronucleus Tests; Microsomes; Mutagenicity Tests; Mutagens; Oils, Volatile; Origanum; Salmonella typhimurium
PubMed: 27891531
DOI: 10.1155/2016/3694901 -
American Journal of Physiology. Renal... Oct 2015Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin...
Heme oxygenase (HO) is a renoprotective protein in the microsome that degrades heme and produces biliverdin. Biliverdin is then reduced to a potent antioxidant bilirubin by biliverdin reductase in the cytosol. Because HO activity does not necessarily correlate with HO mRNA or protein levels, a reliable assay is needed to determine HO activity. Spectrophotometric measurement is tedious and requires a relatively large amount of kidney samples. Moreover, bilirubin is unstable and spontaneously oxidized to biliverdin in vitro. We developed a novel and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify biliverdin to measure HO activity in mice. Biliverdin and its internal standard, a deuterated biliverdin-d4, have MS/MS fragments with m/z transitions of 583 to 297 and 587 to 299, respectively. We prepared lysates of mouse kidneys, and added excess hemin, NADPH, and bilirubin oxidase to convert all bilirubin produced to biliverdin. After 30-min incubation at 37 or 4°C, the samples were analyzed by LC-MS/MS. The difference in the amount of biliverdin between the two temperatures is HO activity. Treating mice with cobalt protoporphyrin, which induces the expression of HO, increased HO activity as determined by biliverdin production. Measuring the production of biliverdin using LC-MS/MS is a more sensitive and specific way to determine HO activity than the spectrophotometric method and allows the detection of subtle changes in renal or other HO activity.
Topics: Animals; Bilirubin; Biliverdine; Calibration; Chromatography, High Pressure Liquid; Heme Oxygenase (Decyclizing); Mice; Mice, Inbred C57BL; Microsomes; Tandem Mass Spectrometry
PubMed: 26224716
DOI: 10.1152/ajprenal.00210.2015