Liver Membranes Research Articles

Acetaminophen Has Lipid Composition-Dependent Membrane Interactivity That Could Be Related to Nephrotoxicity but Not to Analgesic Activity and Hepatotoxicity

Objective: Although acetaminophen is one of the most widely used over-the-counter drugs, the mechanisms by which this classical drug exerts analgesic, hepatotoxic, and nephrotoxic effects remain unclear. We hypothesized that acetaminophen might act on cellular membranes of nerves, liver, and kidneys. In order to verify this hypothesis, we studied the interactivity of acetaminophen with biomimetic lipid bilayer membranes by comparing with structurally related phenacetin. Methods: Liposomal membranes (unilamellar vesicles suspended in the buffer of pH 7.4) were prepared with phospholipids and cholesterol to mimic the membrane lipid composition of neuronal cells, hepatocytes, and nephrocytes. They were subjected to reactions with acetaminophen and phenacetin at clinically relevant concentrations, followed by measuring fluorescence polarization to determine their membrane interactivity to modify membrane fluidity. Results: Acetaminophen and phenacetin interacted with neuro-mimetic and hepato-mimetic membranes to increase membrane fluidity at 10–100 μM. Both drugs were more effective in fluidizing hepato-mimetic membranes than neuro-mimetic membranes. Although the relative membrane-interacting potency was phenacetin >> acetaminophen in neuro-mimetic and hepato-mimetic membranes, such membrane effects conflicted with their relative analgesic and hepatotoxic effects. Acetaminophen and phenacetin strongly interacted with nephro-mimetic membranes to increase membrane fluidity at 2–100 μM and 0.1–100 μM, respectively. Phenacetin interacted significantly with nephro-mimetic membranes at lower concentrations (<2 μM) than acetaminophen, which was consistent with their relative nephrotoxic effects. Conclusion: In comparison with phenacetin, lipid composition-dependent membrane interactivity of acetaminophen could be related to nephrotoxicity but not to analgesic activity and hepatotoxicity.

Toxicological impact of Zinc Nano Particles on tilapia fish (Oreochromis mossambicus)

In this study we investigated the acute toxicity of Zinc Nano Particles (ZnO NPs) and bulk ZnO on tilapia fish (Oreochromis mossambicus). Oreochromis mossambicus was exposed to the different concentration of ZnO NPs, ZnO and mixed solution of both ZnO NPs and ZnO (20 ppb, 20 ppb, 20 ppb) respectively for 96 h. A very high impact was recorded in hematological parameters which shows significant increased (p < 0.05) in count of white WBCs and platelets in all the experimental groups compared tocontrol group. The count of RBCs, Hb, hematocrit and MCHC were significantly decreased. The remarkable changes which were recorded during this study were histopathological lesions in the gills of exposed fish including, disorganization of gill lamella, cartilaginous core disruption, lifting of epithelium, loss of secondary gill lamellae, blood congestion, fusion of secondary gills lamellae, shortening of secondary gills lamellae, atrophy and curling. Disassembly were seen in plasma membrane of liver along with blood congestion, pyknosis, necrosis, hyperplasia and formation of vacuoles. Intestinal alterations which were observed include shortening of villi, necrosis, detachment and fusion of villi and extreme goblet cells formation. It is concluded from the present study that high level of ZnO NPs, ZnO and mixed solution has a strong tendency to alter hematological parameters, histological architecture, therefore, the indiscriminate use of ZnO NPs and ZnO can subsidize in reducing the population of Oreochromis mossambicus in natural water bodies.

AMELIORATIVE ROLE OF GARLIC (ALLIUM SATIVUM) ON CHROMIUM (VI) - INDUCED MEMBRANE DAMAGE IN MALE ALBINO RATS

Objective: Membrane damage is one of the important consequences of chromium (Cr) induced cytotoxicity. Garlic possesses antioxidant property to scavenge the toxic radicals and cytoprotective activity. The aim of the present study is to evaluate the ameliorative role of aqueous extract of garlic (AEG) on Cr-induced membrane damage of both liver and kidneys in male albino rats.&#x0D; Methods: Male albino rats of Wistar strain (80-100 g) were used for the present study. Rats were divided into three groups of almost equal average body weight. The animals of two groups were injected K2Cr2O7 at a dose of 0.8 mg per 100 g body weight per day for 28 days. The animals of one of the Cr-treated groups served as the supplemented group supplied aqueous extract of garlic (AEG) (250 mg per kg body weight daily at an interval of 6 h after injection of Cr for a period of 28 days). The animals of the remaining group received only the vehicle (0.9% NaCl), served as control. The body weights of the animals were taken in each day of treatment schedule.&#x0D; Results: The results indicated that significant increases in membrane cholesterol level as well as significant decreases in membrane phospholipid level in Cr exposed animals suggest structural alterations in both liver and kidneys plasma membrane. Alkaline phosphatase (ALP), total ATPase, and Na+-K+- ATPase activities of plasma membrane were significantly decreased in both liver and kidneys after Cr treatment. On the other hand, AEG supplementation plays a vital role to restore such alterations induced by Cr in plasma membrane of both liver and kidney.&#x0D; Conclusion: These findings indicate that Cr treatment at the present dose and duration induces structural and functional alterations in the plasma membrane in both liver and kidney. However, AEG supplementation restored those alterations induced by Cr in plasma membrane of both liver and kidneys but was not able to eliminate the deposited Cr from the liver and kidney tissues.

Investigation of phytochemical profile and in vivo anti-proliferative effect of Laetiporus versisporus (Lloyd) Imazeki mushroom against diethylnitrosamine-induced hepatocellular carcinoma

The purpose of the study is to explore the bioactive compounds present in Laetiporus versisporus (LVEE) Lloyd Imazeki ethanolic extract and its anticancer activity against hepatocellular carcinoma (HCC) induced by DEN in rats. The bioactive compounds present in the LVEE was analyzed by GC–MS. The anticancer activity of LVEE was analyzed with the DEN induced rats were orally treated with LVEE (250 mg/kg), cyclophosphamide (50 mg/kg) to the respective treatment groups for 45 days. The levels of tissue markers, phase-I metabolizing enzymes was measured calorimetrically. ELISA was used to determine the level of AFP and CEA. The expression of apoptotic genes Bcl-2, p53, caspase 3 and caspase 9 was assessed by real time RT-PCR. Results of the phytochemical screening analysis showed that 12 bioactive molecules were existing in LVEE. Results of pharmacodynamics analysis showed that the 45 days of treatment with LVEE and cyclophosphamide (CPA) therapy significantly (p < 0.05) decreased the levels of MDA, AFP, CEA, cytochrome P450 and cytochrome b5, and increased levels of tissue markers and bilirubin in serum. The findings of RT-PCR analysis showed that the LVEE and CPA were significantly (p < 0.05) downregulated the pro-apoptotic genes Bcl-2 and upregulated the p53, caspase 3 and caspase 9 mRNA expression through mechanisms involved in promoting the cell apoptosis, and preserving the functional integrity of the liver membrane. L. versisporus (LV) can be considered as new anticancer agent that offers new opportunities for anticancer therapy.

Situs Inversus &amp; Adult Polycystic Kidney Disease

Situs Inversus totalis is rare disorder in which there is transposition of organs position. Polycystic kidney disease is a disorder in which water containing cysts are formed in kidneys. Though named as polycystic kidney disease but cysts are formed in other organs such as liver, pancreas, seminal vesicles & arachnoid membrane. Association of these two conditions is rare.

Hepatic and renal expression of Oatp1 in obstructive uropathy. First detection of Oatp1 in urine, a potential biomarker.

Obstructive renal diseases affect renal function and kidney integrity. Nevertheless, little is known about its systemic or extra-renal effects. The organic anion transporting polypeptide 1 (Oatp1) is a carrier expressed in liver and kidneys. In this study, the hepatic and renal expression of Oatp1 was evaluated in rats with obstructive nephropathy. Moreover, the urinary excretion of Oatp1 (Oatp1u ) was evaluated as a potential biomarker for this pathology. Male Wistar rats with bilateral ureteral obstruction for 5hours (BUO5), 24hours (BUO24) or sham operated were used. After 24hours of ureteral releasing, liver and kidney functional parameters, histopathology, Oatp1 tissular expression and Oatp1u were evaluated. For Oatp1u evaluation two groups were added; BUO1 and BUO2 (1 and 2hours of ureteral obstruction, respectively). Both liver and kidney functional parameters and histopathological studies showed alterations in BUO5 and BUO24. In hepatic homogenates, Oatp1 significantly decreased in BUO groups and in total liver membranes no modifications were observed. In renal homogenates, Oatp1 significantly decreased in BUO groups, but in apical kidney membranes, its expression was increased. Oatp1u was only detected in BUO groups, even in those (BUO1, BUO2) in which no alterations in the traditional parameters of renal function were observed. Modulations in liver and renal expression of Oatp1 could be an organism strategy to attenuate the effects of the disease and an attempt to maintain the complex organ cross-talk between liver and kidneys. Oatp1u could be a new, early and specific biomarker of obstructive nephropathy.

How adiponectin fights diabetes

Signal Transduction Adiponectin, a hormone produced in adipose tissue, may provide a useful therapeutic lead for type 2 diabetes because it improves glucose homeostasis and tissue sensitivity to insulin. But how it does so is unclear. Li et al. show that in mice with insulin resistance caused by a high-fat diet, treatment with adiponectin reduced the amount of triglyceride in the liver and in skeletal muscle. It did so by increasing lipoprotein lipase activity and triglyceride uptake into epididymal white adipose tissue and increasing fatty acid oxidation in muscle. These effects decreased the amount of diacylglycerol in the plasma membrane of liver and muscle cells, thus decreasing the activity of protein kinase C family members that impair signaling by the insulin receptor. Proc. Natl. Acad. Sci. U.S.A. 117 , 32584 (2020).

125I-Angiotensin 1-7 binds to a different site than angiotensin 1-7 in tissue membrane preparations.

To study the receptor for Angiotensin (Ang) 1-7 using a radioligand (125I-Ang 1-7)-binding assay. For more than a decade, Mas has been viewed as the receptor for Ang 1-7; however, Ang 1-7 binding has not been pharmacologically characterized in tissue membrane preparations. Radioligand-binding assays were carried out using tissue membrane preparations using radioiodinated Angiotensin 1-7 (125I-Ang 1-7) to characterize its binding site. Non-radioactive 127I-Ang 1-7 was used to test if the addition of an iodine to the tyrosine4 moiety of Ang 1-7 changes the ability of Ang 1-7 to competitively inhibit 125I-Ang 1-7 binding. 125I-Ang 1-7 binds saturably, with moderately high affinity (10-20 nM) to a binding site in rat liver membranes that is displaceable by 127I-Ang 1-7 at nanomolar concentrations (IC50 = 62 nM) while Ang 1-7 displaces at micromolar concentrations (IC50 = 80 µM) at ~22 °C. This binding was also displaceable by inhibitors of metalloproteases at room temperature. This suggests that 125I-Ang 1-7 binds to MMPs and/or ADAMs as well as other liver membrane elements at ~ 22 °C. However, when 125I-Ang 1-7-binding assays were run at 0-4 °C, the same MMP inhibitors did not effectively compete for 125I-Ang 1-7. The addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the binding characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors.

2,3,5,4'-Tetrahydroxystibane-2-O-β-D-glucoside induces liver injury by disrupting bile acid homeostasis and phospholipids efflux

Polygonum multiflorum is a traditional Chinese herbal medicine and has many biological activities such as hair-blacking, anti-atherosclerosis, anti-inflammatory and anti-aging. However, the liver injury induced by P. multiflorum has aroused wide attention in recent years. 2,3,5,4'-tetrahydroxystibane-2-O-β-D-glucoside(TSG) is a main component of P. multiflorum, but the role of TSG in inducing liver injury is unclear. The aim of present study was to evaluate TSG's potential liver injury and effects on bile acid homeostasis and phospholipids efflux. C57 BL/6 J mice received intraperitoneal administration of 400 mg·kg~(-1) of TSG daily for 15 days, and then biochemical indexes of liver injury and changes of phospholipid content were detected. The changes of bile acid compositions were detected by LC-MS/MS. The results showed TSG 400 mg·kg~(-1) significantly increased the content of serum total bile acid(TBA) and alkaline phosphatase(ALP). Elevated free bile acid levels were observed in TSG-treated groups, including β-muricholic acid(β-MCA), ursodeoxycholic acid(UDCA), hyodeoxycholic acid(HDCA), chenodeoxycholic acid(CDCA), deoxcholic acid(DCA) in serum and β-MCA, CDCA in liver. TSG inhibited the protein expression of farnesoid X receptor(FXR) and down stream bile salt export pump(BSEP), which may result in the accumulation of bile acid. TSG also inhibited the expression of 25-hydroxycholesterol-7 alpha-hydroxylase(CYP7 B1), which may disturb the alternative pathway for bile acid synthesis. In addition, intraperitoneal injection of TSG 400 mg·kg~(-1) significantly decreased the content of phospholipids in bile. The research showed that TSG significantly inhibited the expression of multidrug resistance protein 2(MDR2) and destroyed the regular distribution of MDR2 on the bile duct membrane of liver. In vitro results showed that the IC_(50) of TSG on HepG2 cells was about 1 500 μmol·L~(-1) and TSG at 500 μmol·L~(-1)(for 24 h) could destroy the distribution of MDR2 on the bile duct membrane of liver. In conclusion, TSG induced liver injury by disrupting bile acid homeostasis and phospholipids efflux.

Structure and Function of Hepatobiliary ATP Binding Cassette Transporters.

The liver is beyond any doubt the most important metabolic organ of the human body. This function requires an intensive crosstalk within liver cellular structures, but also with other organs. Membrane transport proteins are therefore of upmost importance as they represent the sensors and mediators that shuttle signals from outside to the inside of liver cells and/or vice versa. In this review, we summarize the known literature of liver transport proteins with a clear emphasis on functional and structural information on ATP binding cassette (ABC) transporters, which are expressed in the human liver. These primary active membrane transporters form one of the largest families of membrane proteins. In the liver, they play an essential role in for example bile formation or xenobiotic export. Our review provides a state of the art and comprehensive summary of the current knowledge of hepatobiliary ABC transporters. Clearly, our knowledge has improved with a breath-taking speed over the last few years and will expand further. Thus, this review will provide the status quo and will lay the foundation for new and exciting avenues in liver membrane transporter research.

Antigenic distribution, pathological changes, antibody response and serological detection in non‐pregnant goats following experimental infection by Brucella melitensis

This study describes the pathological changes, antibody response, isolation and distribution patterns following exposure of non-pregnant goats to live Brucella melitensis. Eighteen healthy adult female goats were divided into two equal groups. Group 1 was infected via conjunctival sac with 109 cfu/ml of B.melitensis, while Group 2 was similarly exposed to sterile PBS. Serum and swabs from the eyes and vagina were collected at 5-day intervals. On days 15, 30 and 75 post-infection, 3 goats from each group were killed before the conjunctiva, ovary, oviduct, uterine horn, uterine body and vagina, the submandibular, prescapular and supramammary lymph nodes, the mammary gland, liver, spleen, urinary bladder and synovial membranes were collected for bacterial isolation and pathological study. Exposure of non-pregnant goats to B.melitensis did not produce clinical signs and gross lesions but produced mild necrosis and inflammation in the lymph nodes, the organs of reproductive tract, the mammary gland and urinary bladder. In general, microscopic lesions were most severe in the D75 goats, followed by D30 and D15 goats. Brucella melitensis was most frequent and significantly (p<.05) isolated from the D30 (64.4±25.2%) and least from D15 goats (39.3±26.0%) goats. The organs that were most frequently isolated were the uterus, followed by the mammary gland, supramammary lymph node and urinary bladder. Earliest isolation from the ocular swabs was on day 5, while the vaginal swabs were on day 20 post-infection. The antibody response showed first significant (p<.05) increase on day 15 and reached peak on day 45 post-infection, corresponding with the first detection of sero-converter goats by the RBPT at 15days and by the CFT at 40days post-infection. In conclusion, infected non-pregnant goats shed B.melitensis through the vagina by day 20. The sero-positive goats were detectable by RBPT after 15days but by CFT after 40days. Since both serological tests detected positive goats at different time period of infection, paired-serum samplings might reduce this discrepancy.

Foliar application of zinc and selenium alleviates cadmium and lead toxicity of water spinach – Bioavailability/cytotoxicity study with human cell lines

The present study investigated the effects of foliar application of zinc (Zn) and selenium (Se) on bioavailability of Zn and Se and toxicity of cadmium (Cd) and lead (Pb) to different water spinach ecotypes (LA and HA) grown in slightly (XZ) or moderately (LJY) contaminated fields via in vitro digestion combined with Caco-2/HL-7702 cell model. The obtained results revealed that foliar application of Zn and Se promoted yield, increased total, bioaccessible and bioavailable fractions of Zn and Se in plants, indicating that foliar application is a feasible way of biofortification. Although there was no significant effect on liver cell proliferation (MTT), membrane stability (LDH) and hepatocyte enzyme (ALT and AST) activities, the obvious ecotype and soil dependent fluctuations of lipid peroxidation (MDA) and antioxidant enzyme (SOD, POD and CAT) activities in serum highly suggest that the low accumulator and clean field should be used in agricultural production rather than the high accumulator and contaminated farmland. Moreover, foliar application of Zn and Se improved nutritional quality of all water spinach genotypes in both fields, including increased Fe, vitamin C, cellulose and chlorophyll, maintained concentrations of potassium (K), manganese (Mn), copper (Cu), protein, and nitrate. These results demonstrate that this agricultural management practice may prove to be an effective approach for minimizing health risk and alleviating “hidden hunger” in the developing countries.

MicroRNAs Modulate Drug Resistance-Related Mechanisms in Hepatocellular Carcinoma

Primary liver cancer [hepatocellular carcinoma (HCC)] is one of the most common malignant tumors worldwide, causing serious health threats because of its high morbidity and mortality, rapid growth, and strong invasiveness. Patients with HCC frequently develop resistance to the current chemotherapeutic drugs, and this is largely attributed to the high-level heterogeneity of the tumor tissue. MicroRNAs (miRNAs) are a group of master regulators for multiple physiological and pathological processes and play important roles in the tumorigenesis. More recent studies have indicated that miRNAs also play a non-negligible role in the development of drug resistance in liver cancer. In this review, we summarize the data from the latest studies on the mechanisms of drug resistance in liver cancer, including autophagy, membrane transporters, epithelial–mesenchymal transitions (EMTs), tumor microenvironment, and genes and proteins that are associated with apoptosis. The data herein will provide valuable information for the development of novel approaches to tackle drug resistance in the management of liver cancer.

125‐I‐Sarcosine(1), D‐Alanine(8) Angiotensin II as a Biased Radioligand for AT1 Angiotensin II Receptors

The concept of biased agonism of G protein‐coupled receptors arose from studies of the AT1 subtype of angiotensin (Ang) II receptor. A putative antagonist ligand, sarcosine1, isoleucine4, isoleucine8 Ang II (SII) showed negligible ability to activate G protein signaling pathways of the AT1 receptor, but was able to cause AT1 receptor internalization and mitogen activated protein (MAP) kinase activation at micromolar concentrations. Subsequently, a different Ang II congener; sarcosine1, D‐alanine8 Ang II was developed as a potential therapeutic (TRV‐027; TRV120027) for treatment of acute heart failure. While clinical trials did not support a therapeutic benefit of sarcosine1, D‐alanine8 Ang II for treatment of heart failure, it still promises to be a better biased agonist than SII, which acts only at micromolar concentrations. To assess the potential use of sarcosine1, D‐alanine8 Ang II as a radioligand for quantification of Ang II receptor binding, we radiolabeled sarcosine1, D‐alanine8 Ang II with 125iodine and compared its binding to that of 125I‐sarcosine1 isoleucine8 Ang II in rat liver membranes, which express high levels of AT1 receptors and negligible levels of AT2 receptors. At concentrations up to 5.6 nM, 125I‐sarcosine1, D‐alanine8 Ang II showed little evidence of saturable binding to AT1 receptors, in contrast to 125I‐sarcosine1 isoleucine8 Ang II, which showed a KD of 0.6 nM. At 5.6 nM 125I‐sarcosine1 isoleucine8 Ang II binding to AT1 receptors was 8.5 times greater than 125I‐sarcosine1, D‐alanine8 Ang II. To verify that sarcosine1, D‐alanine8 Ang II binds to liver membrane AT1 receptors, we compared the ability of sarcosine1, D‐alanine8 Ang II and Ang II to compete for 125I‐sarcosine1 isoleucine8 Ang II binding. Sarcosine1 isoleucine8 Ang II and Ang II were similar in their ability to compete for AT1 receptor binding: 6.7±0.7 nM and 15.6±0.8 nM and 84.3±1.1 and 86.7±0.3 % of total binding (IC50, n=2±SD), respectively. This suggests that iodination of tyr4 of sarcosine1, D‐alanine8 Ang II alters its ability to bind to AT1 receptors and potentially act as a biased agonist. Continuing studies will examine the ability of higher concentrations of 125/127I‐sarcosine1, D‐alanine8 Ang II to interact with Ang II receptors.Support or Funding InformationNova Southeastern University President’s Faculty Research Development Grant, Cardiovascular Neuroscience Research Fund

PGRMC1 Stabilizes Cytochromes P450 And Is Required for Activity In Vivo

Progesterone receptor membrane component 1 (PGRMC1) is a membrane‐bound hemoprotein with many ascribed functions that binds cytochromes P450 in yeast and human cells. In this study, we generated a whole‐body Pgrmc1 KO mouse to examine the function of PGRMC1 in cytochrome P450 biology. Here, we report that Pgrmc1 is a novel post‐translational stabilizer of cytochromes P450 required for cytochrome P450 activity in vivo. In mouse liver, Pgrmc1 bound 13 cytochromes P450 of diverse families (Cyp1, 2, 3, 8, and 51) and functioned to maintain liver cytochrome P450 levels post‐transcriptionally. In the absence of Pgrmc1, cytochrome P450 protein levels were 22–70% of wild‐type. These reductions in P450 enzymes were shown to be physiologically important. Pgrmc1 promoted Cyp1a2 activity in vitro as the Vmax for the Cyp1a2‐dependent O‐deethylation of 7‐ethoxycoumarin was 40% lower in the absence of Pgrmc1 in liver membranes. Mechanistically, PGRMC1 stabilized CYP1A2 post‐translationally, doubling the half‐life of the cytochrome P450 in transfected human cells. The stabilization of cytochromes P450 and promotion of cytochrome P450 activity by Pgrmc1 were the result of a protein‐protein interaction between Pgrmc1 and the cytochrome P450 that did not require Pgrmc1 to bind heme. Reductions in Cyp2e1 protein in Pgrmc1 KO mice protected animals against hepatotoxicity during Cyp2e1‐dependent acetaminophen overdose, the leading cause of acute liver failure in the U.S. This study establishes PGRMC1 as a central component controlling cytochrome P450 activity thus making PGRMC1 the first new player in the liver cytochrome P450 system in 50 years as it joins cytochrome P450 oxidoreductase and cytochrome b5. This study also establishes PGRMC1 as a clinically relevant effector of cytochrome P450 activity, which has direct clinical implications related to the efficacy of pharmacotherapies and will be of interest to pharmacologists, clinicians, and the pharmaceutical industry.Support or Funding InformationM.M. was supported by NIH 5T32GM007445, NIH F31HL131185, and American Heart Association 15PRE25250008. P.E. was supported by NIH R01HL077588 and NIH R21HL094774.

Preliminary Characterization of the 125I‐Angiotensin 1‐7 Binding Site in Rodent Liver

Angiotensin 1–7 (Ang 1‐7) is a product of the renin‐angiotensin system that acts centrally and peripherally to reduce blood pressure and improve baroreflex sensitivity. For more than a decade Mas has been viewed as the receptor for Ang 1‐7, however, Ang 1‐7 binding has not been pharmacologically characterized in tissue membrane preparations. In this study, we show that 125I‐Ang 1‐7 binds to a site for which Ang 1‐7 has low affinity. To assess the possibility that the low affinity of Ang 1‐7 to compete for 125/127I‐Ang 1‐7 binding was due to its susceptibility to metabolism by peptidases in the tissue membrane homogenate, the integrity of Ang 1‐7 as well as 127I‐Ang 1‐7 (iodoAng 1‐7) was evaluated by HPLC analysis. Both Ang 1‐7 and iodoAng 1‐7 were substantially metabolized in liver membrane homogenates at room temperature (RT). However, when incubated on ice (~0 C), 125I‐Ang 1‐7 binds with moderately higher affinity (~3 nM versus 10–20 nM at RT) to a binding site in rat liver membranes that is displaceable by iodoAng 1‐7 at nanomolar concentrations, but Ang 1‐7 still shows a low affinity to compete (1.4μM for Ang 1‐7and 69.4nM for iodoAng 1‐7). At RT 125I‐Ang 1‐7 binding is partially displaceable by metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors, e.g., marimastat, batimastat, and GM‐6001 at nanomolar IC50 values, similar to their IC50 values for MMP activity, suggesting that 125I‐Ang 1‐7 binds to MMPs and/or ADAMs as well as other tissue elements at RT. However, when incubated at ~0 C, the MMP and ADAM inhibitors had micromolar IC50 values, suggesting that 125I‐Ang 1‐7 binding to these peptidases is temperature dependent. Our results continue to suggest that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1‐7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1‐7 receptors. Continuing studies are underway to identify the peptidase(s) or other binding moieties to which 125I‐Ang 1‐7 binds to determine their potential therapeutic importance as druggable targets.

Distinct Regulation of σ 1 Receptor Multimerization by Its Agonists and Antagonists in Transfected Cells and Rat Liver Membranes.

Extensive studies have shown that the σ 1 receptor (σ 1R) interacts with and modulates the activity of multiple proteins with important biological functions. Recent crystal structures of σ 1R as a homotrimer differ from a dimer-tetramer model postulated earlier. It remains inconclusive whether ligand binding regulates σ 1R oligomerization. Here, novel nondenaturing gel methods and mutational analysis were used to examine σ 1R oligomerization. In transfected cells, σ 1R exhibited as multimers, dimers, and monomers. Overall, σ 1R agonists decreased, whereas σ 1R antagonists increased σ 1R multimers, suggesting that agonists and antagonists differentially affect the stability of σ 1R multimers. Endogenous σ 1R in rat liver membranes also showed similar regulation of oligomerization as in cells. Mutations at key residues lining the trimerization interface (Arg119, Asp195, Phe191, Trp136, and Gly91) abolished multimerization without disrupting dimerization. Intriguingly, truncation of the N terminus reduced σ 1R to apparent monomer. These results demonstrate that multiple domains play crucial roles in coordinating high-order quaternary organization of σ 1R. The E102Q σ 1R mutant implicated in juvenile amyotrophic lateral sclerosis formed dimers only, suggesting that dysregulation of σ 1R multimeric assembly may impair its function. Interestingly, oligomerization of σ 1R was pH-dependent and correlated with changes in [3H](+)-pentazocine binding affinity and Bmax Combined with mutational analysis, it is reasoned that σ 1R multimers possess high-affinity and high-capacity [3H](+)-pentazocine binding, whereas monomers likely lack binding. These results suggest that σ 1R may exist in interconvertible oligomeric states in a dynamic equilibrium. Further exploration of ligand-regulated σ 1R multimerization may provide novel approaches to modulate the function of σ 1R and its interacting proteins. SIGNIFICANCE STATEMENT: The σ 1 receptor (σ 1R) modulates the activities of various partner proteins. Recently, crystal structures of σ 1R were elucidated as homotrimers. This study used novel nondenaturing gel methods to examine σ1R oligomerization in transfected cells and rat liver membranes. Overall, agonist binding decreased, whereas antagonist binding increased σ 1R multimers, which comprised trimers and larger units. σ 1R multimers were shown to bind [3H](+)-pentazocine with high affinity and high capacity. Furthermore, mutational analysis revealed a crucial role of its N-terminal domain in σ 1R multimerization.

Beneficial Effects of Low-Calorie-Carbohydrate/High-Agar Diet on Cardiometabolic Disorders Associated with Non-Alcoholic Fatty Liver Disease in Obese Rats.

Energy restriction and low carbohydrate diets are recommended as nutrition therapies to prevent becoming overweight or obese. However, their beneficial effects in non-alcoholic fatty liver disease (NAFLD) are less well investigated. In addition, the effects of the type of polysaccharides incorporated into these diets and their contents have been scarcely studied. Therefore, this study aimed to elucidate whether low-calorie-carbohydrate high-agar diets could improve liver metabolic dysfunction, membrane fluidity, oxidative damage, and endothelial dysfunction in obese rats. Obesity was induced by feeding rats a high-fat diet (HFD) for 10 weeks. The obese rats were then divided into two homogenous groups: the first group was fed low-calorie-carbohydrate/high-agar diet (LCC/HA) and the second continued to consume the HFD for 4 weeks [obese control (Ob-C)]. Normo-ponderal rats were fed a normal diet during the entire study, and were used as the control (N-C). Compared with the Ob-C group, body weight, hepatic lipids, low density lipoproteins cholesterol (C), the non esterified cholesterol/phospholipids ratio, serum transaminases activities, and lipid peroxidation markers (thiobarbituric acid reactive substances and lipid hydroperoxides) were reduced in LCC/HA group (P<0.05). However, the serum concentration of high density lipoproteins-C was enhanced (P<0.05). In addition, we observed improved antioxidant defence and endothelial dysfunction associated with antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase (P<0.05), and nitric oxide level (P<0.05). These findings suggest that hypocaloric diets low in energy and carbohydrates and rich in agar may be beneficial against HFD-induced hepatic steatosis damage, and may be a promising therapeutic strategy to counteract NAFLD development associated with obesity.

Bioelectrodes for evaluating molecular therapeutic and toxicity properties

Abstract Recent progress on material designs merged with nanotechnology and biotechnology strategies has advanced studies of complex biological samples on electrodes for cytochrome P450 (CYP)–driven biocatalytic reactions (e.g. liver membrane fractions, cells, and various organ-specific CYP extracts). In addition, protein engineering of CYP enzymes with their reductase partner in membranes (e.g. baculovirus- or Escherichia coli bacteria–expressed CYP microsomes) and other recombinant strategies (e.g. engineered CYP and reductase fusion domains and site-directed CYP mutagenesis) are promising sustainable approaches for offering abundant sources of CYP enzymes for electrocatalytic applications. The combination of in silico and experimental electroanalytical methods with hyphenated approaches and biological assays can offer early and rigorous profiling of new drugs and specialty chemicals for safe exposure and beneficial use.

Abstract P2027: Preliminary Characterization Of The 125 I-angiotensin 1-7 Binding Site In Rat Liver

Angiotensin 1-7 (Ang 1-7) is a product of the renin-angiotensin system (RAS) that can reduce blood pressure and fibrosis. For more than a decade Mas has been viewed as the receptor for Ang 1-7, however, there are lingering doubts regarding the nature of the interaction between the heptapeptide and Mas. To date, no one has pharmacologically characterized Ang 1-7 binding in tissue membrane preparations. In this study we show that 125 I-Ang 1-7 binds to a site for which Ang 1-7 has low (μmolar) affinity, suggesting that the addition of an iodine molecule to the tyrosine in position 4 of Ang 1-7 drastically changes the characteristics of this peptide making it unsuitable for characterization of Ang 1-7 receptors. 125 I-Ang 1-7 binds with moderately high affinity (10-20 nM) and saturability to a binding site in rat liver membranes that is displaceable by 127 I-Ang 1-7 (iodoAng 1-7) at nanomolar concentrations. This binding is also displaceable by inhibitors of metalloproteases. To test the hypothesis that 125 I-Ang 1-7 binds to a peptidase, a series of peptidase inhibitors were evaluated for their ability to inhibit 125 I-Ang 1-7 binding. The non-selective matrix metalloprotease (MMP) and A Disintegrin And Metalloprotease (ADAM) inhibitors marimastat, batimastat, and GM-6001, which inhibit MMP1,2,3,7,8,13,14 and ADAM 9,17, partially inhibited 125 I-Ang 1-7 binding with nanomolar affinities, similar to their IC 50 values for MMPs, suggesting that 125 I-Ang 1-7 binds to MMPs and/or ADAMs as well as other tissue elements. Further testing with the specific MMP-2 (ARP100), MMP-9 and ADAM-17 inhibitors indicate that 125 I-Ang 1-7 is not binding to these peptidases. Continuing studies are underway to establish conditions under which the 125 I-Ang 1-7 ligand is not metabolized by peptidases, while retaining the ability to bind to peptidases is underway to enable the identification of the specific peptidase(s) or other binding moieties to which 125 I-Ang 1-7 binds to determine their potential therapeutic importance as a druggable targets.