In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (±)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 μM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid β-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (?)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 × 10^?4 and 0.177 × 10^?4 mol/mol protein. Multipie dosing increased irreversible binding 3- to 4-fold. © 1993 Wiley-Liss, Inc.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Down-regulation of protein L-isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component beta-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.     

Identification of protein targets for mycophenolic acid acyl glucuronide in rat liver and colon tissue

Covalent binding of acyl glucuronides to proteins is considered an initiating event for the organ toxicity of drugs containing a carboxylic acid group. An acyl glucuronide (AcMPAG) of the immunosuppressant mycophenolic acid was described and shown to form covalent adducts with plasma albumin in vivo. The aim of the present investigation was to identify AcMPAG target proteins in the liver and colon of rats treated with mycophenolate mofetil, which may contribute to a better understanding of the mechanisms responsible for the development of side effects during therapy with this drug. Mycophenolate mofetil was administered per os in to Wistar rats (40 mg/kg/day) over 21 days. Proteins in liver and colon homogenates were separated by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. AcMPAG labeled protein spots were detected by Western blotting. After in-gel tryptic digestion of the protein spots from parallel gels (n = 2), peptides were characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. Data base searching identified AcMPAG target proteins. Tryptic peptides with sufficient signal intensities were subjected to post-source decay analysis. Three proteins in the liver (ATPase/ATP synthase (alpha and beta subunits), protein disulfide isomerase A3 and selenium binding protein) and one protein in the colon (selenium binding protein) were identified as targets for AcMPAG. ATPase/ATP synthase and protein disulfide isomerase are essential proteins involved in the control of the energy and redox state of the cells, whereas the physiological role of selenium binding protein is not fully understood. This study shows for the first time the formation of adducts between tissue proteins and AcMPAG. Whether this chemical modification is associated with compromised protein function and drug toxicity remains to be investigated.     

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the a-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75–78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.     

Myo-inositol enhances teratogenicity of valproic acid in the mouse

BACKGROUND: Valproic acid (VPA) is an anticonvulsant drug that is widely used therapeutically for a variety of neurological conditions. VPA is also well known for its teratogenic potential in both humans and experimental animal models. The typical malformations observed following VPA exposure include neural tube defects (NTDs) and craniofacial and skeletal malformations. Nevertheless, the mechanisms underlying VPA's anticonvulsant efficacy or its teratogenicity remain to be elucidated. It was recently suggested that a relationship exists between VPA exposure and the cellular depletion of myo-inositol (INO). Furthermore, INO has been shown to rescue NTDs in the curly tail mouse. The aim of this study was to investigate the interactions of VPA and INO in the developing embryo. METHODS: For this purpose, 2 strains of mice were used: SWV/Fnn (known to be sensitive to VPA) and LM/Bc (known to be resistant to VPA-induced NTDs). Pregnant females were randomly assigned to 4 experimental groups: control, VPA (600 mg/kg), INO (400 mg/kg), and VPA plus INO. VPA was injected IP at 8.5 days postcoitum (dpc). INO was administered PO twice a day from 6.5 to 10.5 dpc. At term the dams were killed, the uteri were removed, and all of the general toxicological parameters (number of implants, resorptions, dam weight, and fetus weight) were recorded and statistically analyzed. RESULTS: Postimplantation loss in the SWV/Fnn strain and NTDs in the LM/Bc strain were significantly increased after the coadministration of VPA and INO. CONCLUSIONS: This work clearly indicates that INO enhances VPA-induced teratogenicity in the mouse.     

Circadian time-dependent hepatic and renal toxicities to valproic acid in mice

This study aims to investigate whether hepatic and renal valproic acid (VPA) toxicities varied according to the dosing time in the 24-h scale in mice. VPA was administered by i.p. route to different groups of animals at four different circadian stages (1, 7, 13, and 19 h after light onset (HALO)). Biochemical study and histopathological examinations on liver and kidney sections were performed. The results showed that the hepatic and renal toxicity induced by VPA was time related. Animals treated at 19 HALO showed vacuolar degenerative changes, congestions, and inflammatory areas on liver parenchyma. Lesions within proximal tubules were observed in the kidney in groups treated at 19 HALO. The largest increases in alanine aminotransferase, alkaline phosphatase and plasma creatinine activities were also observed at 19 HALO. The obtained data indicate that the optimal hepatic and renal tolerance is observed when VPA was injected in the middle of the light-rest span of mice.     

Lipidomic characteristics and clinical findings of epileptic patients treated with valproic acid

Our early study has found valproic acid (VPA)‐induced lipid dysmetabolism in animal model, however, the details of lipid profiling of VPA‐treated epileptic patients remain unknown. Therefore, in this study, the blood samples of VPA‐treated epileptic patients and VPA‐free controls were collected for lipidomic and biochemical assays. As results, clinical data showed the changes of some blood lipid molecules in VPA‐treated epileptic patients. In lipidomic assays, all 3797 annotated positive ions were identified prior to the data validation. In addition, the number of differentially expressed lipids were identified. And the 133 lipid molecules in VPA‐treated cases were significantly up‐regulated when compared to those in controls, while other 250 lipid metabolites were down‐regulated. Further, these lipid metabolites were mainly constituted with glycerolipids, glycerophopholipids, fatty acyls, sterol lipids. In addition, the most significant elevations of metabolite molecules of triglyceride, sphingomyelin, phosphorylcholine, ceramides, phenolic phthiocerol, as well as topped reductions of phosphoethanolamines, diradylglycerols, 1α,25‐dihydroxy‐24‐oxo‐22‐oxavitamin D3, 2‐deoxy‐20‐hydroxy‐5alpha‐ecdysone 3‐acetate, dolichyl‐4 phosphate were identified respectively. Taken together, these clinical findings demonstrate that negative impacts of exposure to VPA on expression of lipid mediators, progressively disrupting the functions of lipid molecules. Interestingly, these differentially expressed metabolites may be potential biomarkers for screening VPA‐induced dyslipidemia.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

丙戊酸与缺血性脑卒中相关研究进展

动脉粥样硬化和缺血性脑损伤是防治缺血性脑卒中所面临的两大难题,而细胞炎症损伤是它们的共同诱因。丙戊酸作为组蛋白去乙酰化酶抑制剂,具有抑制细胞炎症因子释放及保护神经的作用,所以丙戊酸可能是防治缺血性脑卒中的潜在治疗药物。本文从组蛋白去乙酰化酶对缺血性脑卒中的影响以及丙戊酸的抗炎机制两个方面进行综述。     

Identification of a novel miRNA that increases transient protein expression in combination with valproic acid

Transient gene expression in mammalian cells is an efficient process to produce recombinant proteins for various research applications and large molecule therapeutics development. For the first time, we report a screen to identify human microRNAs (miRNAs) that increase titers after polyethylenimine (PEI) mediated transient transfection of a HEK293 cell line. From a library of 875 miRNAs, we identified 2 miRNAs, miR‐26a‐5p and miR‐337‐5p, that increased human IgG1 (huIgG1) yields by 50 and 25%, respectively. The titer increase was achievable by expressing miR‐26a‐5p from oligonucleotides or a plasmid. Furthermore, combining miR‐26a‐5p with valproic acid (VPA) treatment doubled huIgG1 titers. Assessment of miR‐26a‐5p and VPA treatment across a panel of 32 human and murine antibodies demonstrates that the level of yield enhancement was molecule‐dependent, with most exhibiting a range of 50–100% titer increase. These findings exemplify that combining genetic and chemical manipulation can be an effective strategy to enhance transient transfection productivity. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1139–1145, 2017     

Preliminary studies about novel strategies to reverse chemoresistance to adriamycin regarding glutathione metabolism,peroxisomal and extraperoxisomal hydroperoxide and valproic acid metabolic pathways

Summary— The present work was aimed at defining novel strategies to reverse chemoresistance to anticancer drugs, especially by interfering with cellular glutathione metabolism, peroxisomal and/or extraperoxisomal hydroperoxide metabolic pathways. Preliminary results are presented about molecules we demonstrated to be capable of interfering with hydrogen peroxide metabolism in cells. Prior to describing these molecules, a short overview of glutathione and free radical metabolic pathways is presented as well as a rapid presentation of the characteristics of chemo-sensitivity and -resistance towards the anticancer drug adriamycin, with special emphasis on hydrogen peroxide metabolism. The strategies currently developed to reverse chemoresistance are further presented. in subsequent sections, our own strategy to achieve inhibition of hydrogen peroxide breakdown and stimulation of peroxisomal hydrogen peroxide production is illustrated on the basis of molecular modelling studies and biochemical investigations on extraperoxisomal and peroxisomal metabolic pathways. Preliminary studies on cultured cells have been initiated. The perspective for future studies is presented as well as other possible models of chemoresistance as target for the design of hydrogen peroxide metabolism-interfering pharmacomolecules.     

Phosphorylation of a p38‐like MAPK is involved in sensing cellular redox state and drives atypical tubulin polymer assembly in angiosperms

Reactive oxygen species (ROS) imbalance is a stressful condition for plant cells accompanied by dramatic changes in tubulin cytoskeleton. Here, evidence is provided that alterations in ROS levels directly interfere with the phosphorylation state of a p38‐like MAPK in the angiosperms Triticum turgidum and Arabidopsis thaliana. Both oxidative stress generators and chemicals inducing ROS scavenging or decreasing ROS production resulted in the accumulation of a phospho‐p46 protein similar to p38‐MAPK. Importantly, the rhd2 A. thaliana mutants exhibited a remarkable increase in levels of phospho‐p46. The presence of the p38‐MAPK inhibitor SB203580 attenuated the response to ROS disturbance, prevented microtubule disappearance and resulted in a dramatic decrease in the number of atypical tubulin polymers. Moreover, in roots treated simultaneously with substances inducing ROS overproduction and others resulting in low ROS levels, phospho‐p46 levels and the organization of tubulin cytoskeleton were similar to controls. Collectively, our experimental data suggest, for the first time in plants, that p46 functions as a putative sensor of redox state, the activation of which initiates downstream signalling events leading to microtubule disruption and subsequent assembly of atypical tubulin polymers. Thus, p46 seems to participate in perception of ROS homeostasis disturbance as well as in cellular responses to redox imbalance.     

The action of cytochalasin A on the in vitro polymerization of brain tubulin and muscle G-actin

The presence of cytochalasin A inhibits the self-assembly of beef brain tubulin and rabbit muscle G-actin in vitro and also decreases the colchicine binding of tubulin. Prior reaction of cytochalasin A with 2-mercaptoethanol destroys its inhibitory effects. It is shown that cytochalasin A exerts its actions by reacting with sulfhydryl groups, possibly causing irreversible structural changes in the proteins. Cytochalasin B does not affect the tubulin assembly reaction.     

Polycomb homologs are involved in teratogenicity of valproic acid in mice

BACKGROUND: Valproic acid (VPA) is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its teratogenic mechanisms are unknown. We have attempted to describe a fundamental role of the Polycomb group (Pc-G) in VPA-induced transformations of the axial skeleton. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of vehicle or VPA (800 mg/kg) on gestation day (GD) 8. The expression of genes encoding Polycomb and trithorax groups was measured by quantitative real-time RT-PCR using total RNA isolated from the embryos exposed to vehicle or VPA for 1, 3, and 6 hr. In addition, the use of two less teratogenic antiepileptic chemicals valpromide (VPD) and valnoctamide (VCD) provide reliable evidence to support the relationship between VPA teratogenicity and the Polycomb group. RESULTS: At a teratogenic level, VPA inhibits the expression of the Polycomb group genes, including Eed, Ezh2, Zfp144, Bmi1, Cbx2, Rnf2, and YY1 in the mouse embryos. In contrast, neither VPD nor VCD have significant effects on the expression of those genes affected by VPA. The trithorax group (trx-G) gene MLL, which is known to be required to maintain homeobox gene expression such as the Polycomb gene, is not affected by a teratogenic dose of VPA. CONCLUSIONS: We propose that, during embryonic development, VPA may affect the gene silencing pathway mediated by the Polycomb group complex. The epigenetic mechanism of VPA teratogenicity on anteroposterior patterning is suspected.     

Regulation by thyroid hormone of microtubule assembly and neuronal differentiation

In this review we examine successively: 1) the major effects of thyroid hormone deficiency seen during brain development with special emphasis on the changes in neuronal morphology and migration occurring postnatally in the cerebellum. 2) The effects of this hormone on microtubule assembly during neurite outgrowth and acquisition of neuronal polarity. 3) The changes in expression of the different tubulin isoforms occurring during development in the normal and hypothyroid rat brain. 4) The regulation by thyroid hormone of the transition occurring during development between the juvenile and adult microtubule-associated protein Tau.Special issue dedicated to Dr. Louis Sokoloff.     

Modulation of angiogenesis-related protein synthesis by valproic acid

Recent studies have attested to the antiangiogenic effects of HDAC inhibitors on solid human tumors. The HDAC inhibitor butyrate has been reported to impair tumor-cell-induced angiogenesis. However, due to its poor bioavailability in vivo, the therapeutic use of butyrate is limited. On the other hand, valproic acid has inhibitory effects on carcinoma cells, is known to be well tolerated, and has an excellent bioavailability. We therefore set out to investigate whether the HDAC inhibitor valproic acid also impairs angiogenesis. Our findings indicate that valproic acid represses the relevant angiogenic factors VEGF and FGF in Caco-2 cells. Both, protein expression as well as mRNA levels of VEGF, were reduced to a similar degree. Suppression of ubiquitin-proteasome activity could be a possible reason for valproic acid effects on regulatory angiogenesis proteins. These results suggest that the HDAC inhibitor valproic acid could become a valuable new addition in the attempt to develop alternative therapeutic approaches in the treatment of colon carcinomas.     

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.