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J147 is a novel drug candidate developed to treat neurological dysfunction. Numerous studies have demonstrated the beneficial effects of J147 in cellular and animal models of disease which has led to the transitioning of the compound into human clinical trials. However, no biomarkers for its target engagement have been identified. Here, we determined if specific metabolites in the plasma could be indicative of J147's activity in vivo. Plasma lipidomics data from three independent rodent studies were assessed along with liver lipidomics data from one of the studies. J147 consistently reduced plasma free fatty acid (FFA) levels across the independent studies. Decreased FFA levels were also found in the livers of J147-treated mice that correlated well with those in the plasma. These changes in the liver were associated with activation of the AMP-activated protein kinase/acetyl-CoA carboxylase 1 signaling pathway. A reduction in FFA levels by J147 was confirmed in HepG2 cells, where activation of the AMPK/ACC1 pathway was seen along with increases in acetyl-CoA and ATP levels which correlated with enhanced cellular bioenergetics. Our data show that J147 targets liver cells to activate the AMPK/ACC1 signaling pathway and preserve energy at the expense of inhibiting FFA synthesis., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

During alcoholic fermentation, many parameters, including the nitrogen composition of the must, can affect aroma production. The aim of this study was to examine the impact of several types of nitrogen sources added at different times during fermentation. Nitrogen was added as ammonium or a mixture of amino acids at the beginning of fermentation or at the start of the stationary phase. These conditions were tested with two Saccharomyces cerevisiae strains that have different nitrogen requirements. The additions systematically reduced the fermentation duration. The aroma production was impacted by both the timing of the addition and the composition of the nitrogen source. Propanol appeared to be a metabolic marker of the presence of assimilable nitrogen in the must. The production of ethyl esters was slightly higher after the addition of any type of nitrogen; the production of higher alcohols other than propanol was unchanged, and acetate esters were overproduced due to the overexpression of the genes ATF1 and ATF2. Finally the parameter affecting the most the synthesis of beneficial aromas was the addition timing: The supply of organic nitrogen at the beginning of the stationary phase was more favorable for the synthesis of beneficial aromas., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aminoglycosides are a group of broad-spectrum antibiotics that have been used in the clinic for almost a century. The rapid spread of bacterial genes coding for aminoglycoside-modifying enzymes has, however, dramatically decreased the utility of aminoglycosides. We have previously reported several aminoglycoside potentiators that work by inhibiting aminoglycoside N-6'-acetyltransferase, one of the most common determinants of aminoglycoside resistance. Among these, prodrugs that combine the structure of an aminoglycoside with that of pantothenate into one molecule are especially promising. We report here a series of cellular studies to investigate the activity and mechanism of action of these prodrugs further. Our results reveal a new aminoglycoside resistance inhibitor, as well as the possibility that these prodrugs are transformed into more than one inhibitor in bacteria. We also report that the onset of the potentiators is rapid. Their low cell cytotoxicity, good stability, and potentiation of various aminoglycosides, against both Gram-positive and Gram-negative bacteria, make them interesting compounds for the development of new drugs., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The profile of essential and non-essential elements was traced in the edible sea hares Aplysia depilans Gmelin, Aplysia fasciata Poiret and Aplysia punctata Cuvier. Manganese (Mn), iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) were identified as the major essential elements. Risk assessment evidenced that the levels of cadmium (Cd) and lead (Pb) did not exceed the maximum limit value established by the European Regulation, the contents of chromium (Cr), nickel (Ni) and arsenic (As) being also below the levels established by the FDA guide. A correlation between trace elements levels and desaturation-elongation indexes of fatty acids was found. While Cd, Se and molybdenum (Mo) seem to promote the desaturation-elongation process involved on the production of C20:4n-6c, Ni, Cr and Fe may potentiate the conversion of C18:3n-3c to C20:5n-3c. Furthermore, cobalt (Co), Ni and Cu appear to decrease Δ9 desaturation index. Besides the suggested biosynthetic switch modulated by trace elements, the nutritional value of the species is further strengthened., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

BACKGROUND Regorafenib (RGF) is the drug of choice for treating hepatic carcinoma (HCC), but the drug has drawbacks due to resistance and associated adverse effects. Thus, it becomes crucial to understand the causal 'map' of the resistance conferred by RGF, so that its clinical potency can be amplified, resulting in enhanced efficacy with reduced adverse effects. Metformin (MTF) has been reported to target NLK (Nemo-like kinase) to inhibit non-small lung cancer cells. Based on the literature, the present investigation was carried out to reveal the effect of RGF and MTF, with an expectation that MTF can synergize therapeutic potential as well reduce chances of resistance. MATERIAL AND METHODS Protein expression of hypoxia inducible factors (HIF)-2α, 30 kDa HIV Tat-interacting protein (TIP30), E-cadherin, N-cadherin, and pAMPK were assessed by Western blot analysis. RGF and MTF were exposed to MHCC97H cell and proliferation was quantified by assay of cell viability. Gene silencing and chromatin immunoprecipitation assay were done to reveal the relationship between TIP30 and HIF-2α. The impact of RGF and MTF together on postoperative recurrence and lung metastasis of hepatocellular carcinoma was investigated using tumor engrafted mice after administration of MTF and RGF once daily for 35 days. Immunohistochemistry was used to reveal CD31, Ki67, and TUNEL. RESULTS The results suggested MTF-RGF combination lowered expression of HIF-2α gene silencing and suggested increased TIP30 after reduction of HIF-2α. The chromatin immunoprecipitation study indicated that under hypoxia, HIF-2α could bind with TIP30 promoter. Cell number quantification (CCK8), viable cell count, and apoptosis data (using Annexin V-FITC) indicated co-administration of RGF and MTF reduced cell proliferation, encouraging cell apoptosis, and reduced epithelial-mesenchymal transition course. Thus, in orthotopic mice, the RGF-MTF combination exhibited substantial reduction of HCC in lung metastasis and postoperative relapse. CONCLUSIONS MTF can enhance the potential of RGF and inhibit the recurrence and metastasis of HCC after postoperative liver section by regulating the levels of TIP30 and HIF-2α.

Stimulation of vascular smooth muscle (VSM) α1-adrenoceptors induces myosin phosphorylation and vasoconstriction via mobilisation of intracellular calcium and production of specific eicosanoids. Polyunsaturated fatty acid (PUFA) biosynthesis in VSM cells is involved, although the precise mechanism is not known. To address this, we characterised PUFA biosynthesis in VSM cells and determined its role in intracellular calcium release and eicosanoid production. Murine VSM cells converted 18:2n-6 to longer chain PUFA including 22:5n-6. Δ6 (D6d) and Δ5 (D5d) desaturase, and elongase (Elovl) 5 were expressed. Elovl2 was not detected in human, mouse or rat VSM cells, or in rat or mouse aortae, but tit was not associated with hypermethylation of its promoter. D6d or D5d inhibition reduced 18:3n-6 and 20:4n-6 synthesis, respectively, and induced concentration-related decrease in phenylephrine-mediated calcium release, and in PGE2 and PGF2α secretion. Together these findings suggest that PUFA biosynthesis in VSM cells is involved in calcium release associated with vasoconstriction., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The antimutagenicity of α-hexylcinnamaldehyde (1), a semisynthetic and more stable derivative of cinnamaldehyde, was evaluated against common environmental pollutants in the bacterial reverse mutation assay. The pre-, co-, and post-treatment protocols were applied to assess the involvement of desmutagenic and/or bioantimutagenic mechanisms. Compound 1 (9-900 μM) produced a strong antimutagenicity (>40% inhibition) in the Salmonella typhimurium TA98 strain against the nitroarenes 2-nitrofluorene and 1-nitropyrene in almost all experimental conditions. A strong inhibition was also reached against the nitroarene 1,8-dinitropyrene and the arylamine 2-aminoanthracene in the cotreatment at the highest concentrations tested. In order to evaluate if an inhibition of bacterial nitroreductase (NR) and O-acetyltransferase (OAT) could be involved in the antimutagenicity of 1 against nitroarenes, the substance was further tested against 1-nitropyrene (activated by both NR and OAT) in TA98NR and TA98 1,8-DNP strains (lacking the NR and OAT enzymes, respectively). Although both desmutagenic and bioantimutagenic mechanisms appear mostly involved in the antimutagenicity of 1, based on data obtained in the TA98NR strain, applying the pretreatment protocol, compound 1 seems to act as an inhibitor of the OAT-mediated mutagen bioactivation. These results provide justification for further studies on 1 as a possible chemopreventive agent.

Although the regulation of taxol biosynthesis at the transcriptional level remains unclear, 10-deacetylbaccatin III-10 β-O-acetyl transferase (DBAT) is a critical enzyme in the biosynthesis of taxol. The 1740 bp fragment 5'-flanking sequence of the dbat gene was cloned from Taxus chinensis cells. Important regulatory elements needed for activity of the dbat promoter were located by deletion analyses in T. chinensis cells. A novel WRKY transcription factor, TcWRKY1, was isolated with the yeast one-hybrid system from a T. chinensis cell cDNA library using the important regulatory elements as bait. The gene expression of TcWRKY1 in T. chinensis suspension cells was specifically induced by methyl jasmonate (MeJA). Biochemical analysis indicated that TcWRKY1 protein specifically interacts with the two W-box (TGAC) cis-elements among the important regulatory elements. Overexpression of TcWRKY1 enhanced dbat expression in T. chinensis suspension cells, and RNA interference (RNAi) reduced the level of transcripts of dbat. These results suggest that TcWRKY1 participates in regulation of taxol biosynthesis in T. chinensis cells, and that dbat is a target gene of this transcription factor. This research also provides a potential candidate gene for engineering increased taxol accumulation in Taxus cell cultures., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Esculetin, an antioxidant, has been used in the treatment of a variety of diseases. This study aimed to investigate the protective effect of esculetin in attenuating streptozotocin (STZ)-induced type I diabetic nephropathy and to understand the molecular mechanism involved in it. Sprague-Dawley rats were rendered diabetic using a single dose of STZ (55 mg/kg, i.p.). Protein expression of PPARγ and transforming growth factor-β1 (TGF-β1) was detected by immunoblotting and immunohistochemistry respectively. RNA expression levels of Mmp13 and Bmp6 were detected by RT-PCR analysis. In diabetic rats, esculetin treatment resulted in a significant decrease in blood glucose, blood urea nitrogen, and plasma creatinine and increase in plasma albumin levels. Esculetin treatment attenuates the downregulation of PPARγ in diabetic kidney, which in turn blocks the TGF-β1-mediated fibronectin expression. In addition, it attenuates the decrease in mono-methylation (K4) and acetylation of histone H3 in diabetic kidney. RT-PCR analysis revealed that esculetin treatment provides protection by decreasing antifibrotic Bmp6 and increasing fibrogenic Mmp13 mRNA expression in diabetic kidney. This is the first report to show that protection observed by esculetin treatment involves alteration in mRNA expression of Mmp13 and Bmp6 genes either directly via altered histone H3 modifications or indirectly by inhibiting the PPARγ/TGF-β1 pathway.

In the CNS oligodendrocytes produce myelin and ensheath individual axons after birth. Demyelination disables saltatory conduction and leads to loss of neural functions. Oligodendrocyte precursor cells (OPCs) are immature and abundant reservoir cells in the adult brain that are capable of differentiating into myelinating oligodendrocytes. Upon demyelination insults, OPCs are spontaneously induced to differentiate in order to remyelinate denuded axons and promote functional recovery. While remyelination is an efficient regenerative process in the CNS, it often fails in the chronic phase of multiple sclerosis (MS). OPCs are nonetheless preserved in many MS lesions, suggesting that arrested OPC differentiation underlies remyelination failure in chronic MS. Understanding the molecular pathology of this arrested differentiation and remyelination failure in chronic MS is critical for developing remyelination medicines that will promote a full functional recovery in these patients. Recently, TIP30 was identified as an inhibitor of OPC differentiation in MS. TIP30 inhibits proper nucleocytoplasmic transport and thus disables nuclear import of transcription factors that are required for differentiation. TIP30 may also increase susceptibility of OPCs to cell death. In this review, we examine the pathophysiological nature of remyelination failure in chronic MS and discuss the role of TIP30 as a novel therapeutic target.

Platelet-activating factor (PAF) is a potent inflammatory mediator, which seems to play a role in the pathogenesis of several AIDS manifestations such as AIDS dementia complex, Kaposi's sarcoma, and HIV-related nephropathy. PAF antagonists have been studied in these conditions with promising results. In order to examine the possible interactions between PAF and antiretroviral therapy, we studied the effect of nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors against PAF biological activities and its basic biosynthetic enzymes dithiothreitol-insensitive PAF-cholinephosphotransferase (PAF-CPT) and lyso-PAF-acetyltransferase (Lyso-PAF-AT), as well as its main degradative enzyme PAF-acetylhydrolase, of human mesangial cell line (HMC). We also studied the effect of several backbones and highly active antiretroviral therapy (HAART) regimens against PAF activity. Among the drugs tested, several inhibited PAF-induced platelet aggregation in a concentration-depended manner, with tenofovir, efavirenz, and ritonavir exhibiting the higher inhibitory effect. In addition, when these drugs were combined in backbones and HAART regimens based on American antiretroviral therapy proposals, they also synergistically exhibited an inhibitory effect against PAF-induced platelet aggregation. Several of these drugs have also inhibited in vitro microsomal PAF-CPT activity, and concentrations of lopinavir-r or tenofovir-DF (similar to their IC(50) against PAF-induced platelet aggregation) exhibited the same effect against PAF-CPT and Lyso-PAF-AT when added in the cell medium of cultured HMC. In addition, in naïve patients treated with one of the most potent anti-PAF HAART regimens (efavirenz/emtricitabine/tenofovir-DF) for a period of 1 month, a significant reduction of the specific activity of PAF-CPT of washed human leukocytes of these patients was also observed, compared with its levels before the HAART treatment. These promising results need to be further studied and confirmed by additional in vivo tests in order to optimize HAART efficacy.

High levels of the pro-inflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), are present in the gut mucosa of patients suffering form various diseases, most notably inflammatory bowel diseases (IBD). Since the inflammatory milieu can cause important alterations in epithelial cell function, we examined the cytokine effects on the expression of the enterocyte differentiation marker, intestinal alkaline phosphatase (IAP), a protein that detoxifies bacterial lipopolysaccharides (LPS) and limits fat absorption. Sodium butyrate (NaBu), a short-chain fatty acid and histone deacetylase (HDAC) inhibitor, was used to induce IAP expression in HT-29 cells and the cells were also treated +/- the cytokines. Northern blots confirmed IAP induction by NaBu, however, pretreatment (6 h) with either cytokine showed a dose-dependent inhibition of IAP expression. IAP Western analyses and alkaline phosphatase enzyme assays corroborated the Northern data and confirmed that the cytokines inhibit IAP induction. Transient transfections with a reporter plasmid carrying the human IAP promoter showed significant inhibition of NaBu-induced IAP gene activation by the cytokines (100 and 60% inhibition with IL-1beta and TNF-alpha, respectively). Western analyses showed that NaBu induced H4 and H3 histone acetylation, and pretreatment with IL-1beta or TNF-alpha did not change this global acetylation pattern. In contrast, chromatin immunoprecipitation showed that local histone acetylation of the IAP promoter region was specifically inhibited by either cytokine. We conclude that IL-1beta and TNF-alpha inhibit NaBu-induced IAP gene expression, likely by blocking the histone acetylation within its promoter. Cytokine-mediated IAP gene silencing may have important implications for gut epithelial function in the setting of intestinal inflammatory conditions.

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, -3, -8, and -9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ';Replication Complexes'' during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/- aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

We have investigated the effect of HER-2 overexpression on resistance to the aromatase inhibitor letrozole in MCF-7 breast cancer cells stably expressing cellular aromatase (MCF-7/CA). MCF-7/CA cells overexpressing HER-2 showed a >2-fold increase in estrogen receptor (ER)-mediated transcriptional reporter activity upon treatment with androstenedione compared with vector-only control MCF-7/CA cells. Co-treatment with letrozole did not abrogate androstenedione-induced transcription and cell proliferation in HER-2-overexpressing cells. Chromatin immunoprecipitation assays using cross-linked protein-DNA from MCF-7/CA/HER-2 cells indicated ligand-independent association of the ERalpha coactivators AIB-1 and CBP to the promoter region of the estrogen-responsive pS2 gene. Upon treatment with androstenedione, there were increased associations of AIB1 and CBP with the pS2 promoter in the HER-2-overexpressing compared with control MCF-7/CA cells. These results suggest that ligand-independent recruitment of coactivator complexes to estrogen-responsive promoters as a result of HER-2 overexpression may play a role in the development of letrozole resistance.

Steroid receptor coactivator-3 (SRC-3/AIB1) is an oncogene frequently amplified and overexpressed in breast cancers. Here we report that SRC-3 interacts with REGgamma, a proteasome activator known to stimulate the trypsin-like activity of the 20S proteasome. RNAi knockdown and gain-of-function experiments suggest that REGgamma promotes SRC-3 protein degradation. Cellular levels of REGgamma expression affect estrogen-receptor target-gene expression and cell growth as a result of its ability to promote degradation of the SRC-3 protein. In vitro proteasome proteolysis assays using purified REGgamma, SRC-3, and the 20S proteasome reinforce these conclusions and demonstrate that REGgamma promotes the degradation of SRC-3 in a ubiquitin- and ATP-independent manner. This work demonstrates the first example of a physiologically relevant endogenous cellular target for the REGgamma-proteasome complex. It also highlights the fact that an alternative mode of proteasome-mediated protein degradation, independent of the 19S proteasome regulatory cap, targets the SRC-3 protein for degradation.

Platelet activating factor (PAF) is a potent inflammatory mediator produced by various renal cells and it is implicated in renal pathology. The aim of this study is the characterization of remodeling lyso-PAF acetyltransferase, which is activated under inflammatory conditions, in human mesangial cell. Total membranes of mesangial cells were isolated and enzymatic activity and kinetic parameters were determined by trichloroacetic acid precipitation method. The effect of BSA, divalent cations, EDTA, and various chemicals on the activity of lyso-PAF acetyltransferase was also studied. Various detergents were also tested for the solubilization of the enzyme and only glycerol did not affect its activity. Partial purification of solubilized enzyme preparations of human kidney tissue and mesangial cells was performed on anion exchange column chromatography and native-PAGE electrophoresis and two active fractions were detected.

The mitochondrial enzyme N-acetylglutamate synthase (NAGS) produces N-acetylglutamate serving as an allosteric activator of carbamylphosphate synthetase 1, the first enzyme of the urea cycle. Autosomal recessively inherited NAGS deficiency (NAGSD) leads to severe neonatal or late-onset hyperammonemia. To date few patients have been described and the gene involved was described only recently. In this study, another three families affected by NAGSD were analyzed for NAGS gene mutations resulting in the identification of three novel missense mutations (C200R [c.598T > C], S410P [c.1228T > C], A518T [c.1552G > A]). In order to investigate the effects of these three and two additional previously published missense mutations on enzyme activity, the mutated proteins were overexpressed in a bacterial expression system using the NAGS deficient E. coli strain NK5992. All mutated proteins showed a severe decrease in enzyme activity providing evidence for the disease-causing nature of the mutations. In addition, we expressed the full-length NAGS wild type protein including the mitochondrial leading sequence, the mature protein as well as a highly conserved core protein. NAGS activity was detected in all three recombinant proteins but varied regarding activity levels and response to stimulation by l-arginine. In conclusion, overexpression of wild type and mutated NAGS proteins in E. coli provides a suitable tool for functional analysis of NAGS deficiency.

The relationship between daily rhythms in GABA content and melatonin (MEL) content, as well as the effect of GABA treatment during either the day time and night time phases on MEL levels and N-acetyltransferase (NAT) activity, were studied in the brains of two insect species, the grasshopper Oedipoda caerulescens and the damselfly Ischnura graellsii. In O. caerulescens, levels of GABA in the optic lobes showed significant daily variation, with a marked increase during the light-to-dark transition period. In contrast, in the brain of I. graellssi, two daily peaks in GABA levels were observed, during the light-to-dark and the dark-to-light transition periods. In both insects the maximal levels of GABA occurred 4-6 h in advance of the nocturnal MEL peak, which was associated with a reduction in GABA levels. In both insects, treatment with GABA (1 microg/microl, intracranial injection) during the night was followed by a significant reduction in melatonin levels and NAT activity. In contrast, GABA administered during the day time increased brain MEL levels and synthesis. These data suggest that GABA acts as a modulator of light/dark-dependent melatonin synthesis in the insect brain.

Mammalian pinealocytes synthesize and secrete melatonin. The synthesis of melatonin is regulated by several biogenic amine, amino acid and peptide transmitters. In our previous study, the delta- and mu-opioid receptors have been identified and characterized in bovine pinealocytes. In order to elaborate the function of different types of opioid receptors in regulating melatonin synthesis, we used a selective mu-opioid receptor agonist, Tyr-[D-Ala(2), N-methyl-phe(4), glycol(5)] (DAMGO), a selective delta-opioid receptor agonist, Enkephalin [D-Pen(2), D-Pen(5)], (DPDPE) and a selective kappa-opioid receptor agonist, ((+)-(5alpha, 7alpha, 8beta)-N-methyl-N-[7- (1-pyrrolidinyl)-1-oxaspiro [4,5] dec-8-yl]-benzene acetamide) (U69593) to investigate the activity of N-acetyltransferase (NAT) activity and melatonin secretion. The results of the present study show that both DAMGO and DPDPE stimulated NAT activity and increased the level of melatonin in cultured bovine pinealocytes. These stimulatory effects were blocked by naloxone, an opioid receptor antagonist. However, the kappa-opioid receptor agonist U69593 was unable to alter either the activity of NAT or the level of melatonin. In order to clarify the mechanism of how the activation of mu- and delta-opioid receptors in bovine pinealocytes leads to an increase in NAT activity, cyclic AMP levels were measured after bovine pinealocytes were treated with morphine, DAMGO and DPDPE. The results indicated that these stimulatory effects acted via induction of cAMP production. This study reveals that the stimulatory effect of opioid receptor on melatonin synthesis is mediated via the activation of adenylate cyclase system.

In eukaryotes, genomic DNA is packaged with histone proteins into the cell nucleus as chromatin, condensing the DNA > 10,000-fold. Chromatin is highly dynamic and exerts profound control on gene expression. Localised chromatin decondensation facilitates access of nuclear machinery. Chromatin displays epigenetic inheritance, in that changes in its structure can pass to the next generation independently of the DNA sequence itself. It is now clear that the post-translational modification of histones, for example, acetylation, methylation and phosphorylation, plays a crucial role in the regulation of nuclear function through the 'histone code'. There has been significant progress in identifying and understanding the enzymes that control these complex processes, in particular histone acetyltransferases and histone deacetylases. The exciting discovery that compounds inhibiting histone deacetylase activity also have antitumour properties has focused attention on their use as anticancer drugs. As a consequence, there is ongoing evaluation of several histone deacetylase inhibitor compounds in Phase I and II clinical trials with promising early results. It is likely that many of the enzymes involved in the control of histone modification will provide therapeutic opportunities for the treatment of cancer, including histone methyltransferases and Aurora kinases.

Background: Tea extracts have antiallergic and anti-inflammatory actions in rats and mice. However the mechanism through which tea polyphenols act in vivo are still incompletely understood. We found inhibitory effects of black tea extracts on an fMLP-induced aggregating response in a rabbit platelet-polymorphonuclear leukocyte (PMN) system., Method: To elucidate whether 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) production in PMNs and/or PAF-stimulated platelet activation were inhibited, the effects of tea polyphenols were investigated on the enzyme activity of acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acetyltransferase (EC 2.3.1.67), PAF biosynthesis in A23187-activated rabbit PMNs, and rabbit platelet aggregation. By comparing the inhibitory effects of 31 galloyl esters and gallic acid, the structure-inhibitory activity relationship was characterized., Results: Theaflavin and its galloyl esters and pentagalloyl glucose were found to be potent inhibitors of the acetyltransferase (IC(50) = 28-58 microM) and the PAF biosynthesis as well as (-)-epicatechin-3-O-gallate (IC(50) = 72 +/- 13 microM) and (-)-epigallocatechin-3-O-gallate (IC(50) = 46 +/- 6 microM). On the other hand, flavan-3-ols without galloyl group at C-3 and gallic acid did not show significant enzyme inhibition. In addition, theaflavin and its galloyl esters (IC(50) = 32-77 microM) and geranyl gallate, farnesyl gallate and geranylgeranyl gallate (IC(50) = 6.4-7.6 microM) were found to be potent inhibitors of PAF- and TPA-induced rabbit platelet aggregation but not A23187-induced aggregation., Conclusions: Theaflavin and its galloyl esters in black tea extract, and isoprenyl gallates were potent inhibitors of PAF synthesis and platelet aggregation and these activities may be relevant to the claimed therapeutic effects of tea extracts., (Copyright 2004 S. Karger AG, Basel)

Trichostatin A (TSA), a histone deacetylase inhibitor, strongly increases acetylation of the N-terminal tails of histone H3. Many studies have correlated the function of TSA with the hyperacetylation of histone. Although histone H3 is known to be phosphorylated, the effect of acetylation on phosphorylation is not known. Here, we report that in JB6 cells, TSA induces both acetylation at lysine 9 and phosphorylation at serine 28 of histone H3. UVB irradiation, which is known to induce phosphorylation at serine 28, did not significantly affect phosphorylation of histone H3 in TSA-pretreated JB6 cells. In contrast, TSA markedly increased phosphorylation and acetylation of histone H3 in UVB-pretreated JB6 cells. TSA strongly activated MAP kinases. Moreover, PD98059 and SB202190 inhibited TSA-induced phosphorylation but not acetylation of histone H3. Dominant negative mutant ERK2 and dominant negative mutant p38 kinase blocked TSA-stimulated phosphorylation of histone H3 at serine 28. The results indicate that TSA-induced phosphorylation of histone H3 at serine 28 occurs through activation of the MAP kinase pathway and phosphorylated histone H3 is more sensitive to TSA-induced hyperacetylation. The facilitation of phosphorylation and acetylation of histone H3 induced by TSA may play a critical regulatory role in chromatin remodeling and gene expression.

Background: The male antifertility agent gossypol has been reported to induce spermidine/spermine N1-acetyltransferase (SSAT) in canine prostate cells. As SSAT is the rate-controlling enzyme in the catabolism of the polyamines and is involved in the development of acute pancreatitis in a recent transgenic rat model, we exposed normal and transgenic rats over-expressing SSAT to gossypol to evaluate its effect on pancreatic polyamine metabolism and organ integrity., Methods: Pancreatic SSAT activity, polyamine pools, pancreatic histology and plasma 2-amylase activity were determined after different doses of gossypol., Results: Gossypol increased pancreatic putrescine and decreased spermidine and spermine pools in normal rats accompanied by tissue oedema and significantly elevated plasma amylase activity. In transgenic rats, the drug strikingly induced SSAT, profoundly depleted the higher polyamines and caused distinct pancreatitis. The combination of gossypol at doses harmless to transgenic pancreas with an inhibitor of polyamine oxidase caused massive synergistic induction of SSAT, profound depletion of the polyamine pools and acute pancreatitis., Conclusions: The results indicate that gossypol induces pancreatitis through an activation of polyamine catabolism.

Sixteen isolates of Enterococcus faecalis were recovered from retail poultry samples (seven chickens and nine turkeys) purchased from grocery stores in the greater Washington, D.C., area. PCR for known streptogramin resistance genes identified vat(E) in five E. faecalis isolates (three isolates from chickens and two isolates from turkeys). The vat(E) gene was transmissible on a ca. 70-kb plasmid, along with resistance to erythromycin, tetracycline, and streptomycin, by conjugation to E. faecalis and Enterococcus faecium recipient strains. DNA sequencing showed little variation between E. faecalis vat(E) genes from the chicken samples; however, one E. faecalis vat(E) gene from a turkey sample possessed 5 nucleotide changes that resulted in four amino acid substitutions. None of these substitutions in the vat(E) allele have previously been described. This is the first report of vat(E) in E. faecalis and its transferability to E. faecium, which indicates that E. faecalis can act as a reservoir for the dissemination of vat(E)-mediated streptogramin resistance to E. faecium.

In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.

We have previously established that PAF-dependent transacetylase (TA) purified to apparent homogeneity from rat kidney membranes and cytosol contains three separate catalytic activities, namely PAF lysophospholipid transacetylase (TAL), PAF sphingosine transacetylase (TAS), and PAF acetylhydrolase (AH). In the present investigation, we studied the biochemical factors and mechanism(s) that differentially regulate these three TA activities of the purified enzymes. We found that only the TAS activity of the TA purified from the membranes was stimulated by phosphatidyl-serine (PS) with optimal concentration of activation occurring at 25 microM. Other acidic phospholipids, such as phosphatidylinositol (PI) and phosphatidylinositol 4-phosphate (PIP), are partially effective, while diacylglycerol and free fatty acids had no effect on the TAS activity. PS exerted its effect on the TAS activity through the increases of both Km and Vmax. In addition, N-ethylmalimide (NEM) and dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) strongly inhibited the TAS activity and partially decreased the TAL and AH activities of the purified membrane enzyme in a dose-dependent manner. The addition of PS, but not by its substrate, sphingosine, could prevented the inhibition by NEM on the basal level of TAS. On the other hand, the inhibition of TAL by NEM and DTNB were partially protected by the substrate, lysoplasmalogens. Furthermore, PAF fully protects the inhibition of AH, partially protects the inhibition of TAL, and does not protect the inhibition of TAS by NEM. These results suggested that the three individual catalytic activities of TA have different dependencies on the thiol-containing residue(s) of the enzyme, i.e., cysteine. Furthermore, the nonresponsiveness of the purified cytosolic TAS to PS activation is consistent with our previous notions that membrane and cytosolic TA are posttranslationally distinct.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase., (Copyright 2000 Wiley-Liss, Inc.)

In comparison with dietary high-linoleate safflower oil, high alpha-linolenate perilla oil decreased alkylacyl- and alkenylacyl-glycerophosphocholine (GPC) content in rat kidney by roughly 30 and 25%, respectively. The fatty acid composition was also modified by high alpha-linolenate oil; arachidonic acid (AA) level in alkylacyl-GPC, a platelet-activating factor (PAF) precursor, decreased by 30% along with concomitant increases in the n-3 fatty acid levels. PAF contents under resting conditions were similarly low in the two dietary groups. Fifteen minutes after endotoxin administration, PAF and lyso-PAF contents increased significantly, and the PAF content in the high alpha-linolenate group was 60% lower than in the high linoleate group; the lyso-PAF contents also tended to be lower. Lyso-PAF acetyltransferase and CoA-independent transacylase activities in kidney microsomes increased significantly after endotoxin administration, while PAF acetylhydrolase activity in the cytosol was relatively unchanged. The lyso-PAF acetyltransferase and PAF acetylhydrolase activities did not differ between the two dietary groups, but the CoA-independent transacylase activity was roughly 30% lower in the high alpha-linolenate group. In agreement with in vitro study, our present study demonstrates that dietary high alpha-linolenate suppresses PAF production in rat kidney during systemic endotoxemia, and which is mainly due to the decrease in alkylacyl-GPC content, altered fatty acid compositions of the precursor lipids and lower CoA-independent transacylase activity.

Numerous novel beta-lactamases and aminoglycoside-modifying enzymes with altered substrate profiles continue to be identified. Plasmid-mediated transmission of many of these enzymes readily occurs due to inclusion of the encoding genes in mobile gene cassettes. Recent crystallographic determinations of the structures of metallo-beta-lactamases and aminoglycoside-modifying enzymes provide the opportunity for the rational design of inhibitors.

The results presented here demonstrate that a cis-diamminedichloroplatinum(II) (DDP)-resistant human ovarian-carcinoma cell line is also cross-resistant to the spermine analogue N1,N12-bis(ethyl)spermine (BESPM). We report that C13* cells, which are approximately 20-fold resistant to DDP, similarly showed 7-fold resistance to BESPM by colony-forming assay with an IC50 value of 24.6 +/- 2 microM vs. 3.4 +/- 0.8 microM of 2008 cells. Resistance appears to be the result of many effects, such as different morphological and functional modifications of mitochondria. Furthermore, although BESPM accumulation was almost identical in sensitive and resistant cells, the intracellular polyamine pool of the 2 cell lines was differentially affected by this polyamine analogue. In fact, when spermidine (SPD) was still detectable in C13* cells, in 2008 cells it was not, and the spermine (SPM) content was always more markedly reduced in sensitive cells than in the resistant variant. The lower polyamine content of 2008 cells could be related to a higher degree of induction of spermidine/ spermine N1-acetyltransferase (SSAT) activity by BESPM in sensitive cells than in their resistant counterpart. Despite the observed cross-resistance, the combination of the 2 drugs resulted in supra-additive and synergistic effects in both cell lines, depending on concentration, as assessed by median-effect analysis of the survival data. The effectiveness of this combination was also confirmed by the increased accumulation of cells in the G2/M phase of the cell cycle in both cell lines. Taken together, these data suggest that BESPM effect on cell growth of DDP-sensitive and DDP-resistant cells involves multiple mechanisms that are differently modulated by the DDP-resistant phenotype.

Objective: To determine the effect of 150 mg/day fluvoxamine on the activities of CYP1A2, CYP2D6, CYP3A, N-acetyltransferase-2 (NAT2), and xanthine oxidase (XO) by phenotyping with caffeine, dextromethorphan, and midazolam., Methods: Oral caffeine (2 mg/kg), oral dextromethorphan (30 mg), and intravenous midazolam (0.025 mg/kg) were administered to 10 white male volunteers every 14 days for 4 months and to 10 white premenopausal female volunteers during the midfollicular and midluteal phases of the menstrual cycle for 4 complete cycles (8 total phenotyping measures). The first 6 phenotyping measures were used to establish baseline activity. Subjects were given 150 mg/day fluvoxamine for the fourth month or cycle of the study. Enzyme activity for CYP1A2, CYP2D6, NAT2, and XO was expressed as urinary metabolite ratios. Midazolam plasma clearance was used to express CYP3A activity., Results: No difference between baseline and weeks 2 and 4 of fluvoxamine therapy was observed for NAT2 or XO metabolite ratios. For CYP1A2, CYP2D6, and CYP3A phenotypes, significant differences existed between baseline and fluvoxamine therapy. For CYP1A2, the mean urinary metabolite ratio (+/-SD) was 7.53 +/- 7.44 at baseline and 4.30 +/- 2.82 with fluvoxamine ( P = .012). Mean CYP2D6 molar urinary dextromethorphan ratios before and after fluvoxamine therapy were 0.00780 +/- 0.00694 and 0.0153 +/- 0.0127, respectively (P = .011). Midazolam clearance decreased from 0.0081 +/ 0.0024 L/min/kg at baseline to 0.0054 +/- 0.0021 L/min/kg with therapy (P = .0091). For CYP1A2, CYP2D6, and CYP3A, fluvoxamine therapy changed the phenotyping measures by a median of -44.4%, 123.5%, and -34.4%, respectively., Conclusions: We concluded that fluvoxamine may cause significant inhibition of CYP1A2, CYP2D6, and CYP3A activity. This metabolic inhibition may have serious implications for a variety medications.

Treatment of cells with sodium butyrate is known to increase histone acetylation by inhibiting deacetylases. Here we have observed, in cultured hepatoma cells, that the potent serine-threonine phosphatase inhibitors, okadaic acid or calyculin A, inhibited phosphatase activity and concomitantly decreased the histone acetylation classically maintained by sodium butyrate. These results suggest that a protein phosphatase may mediate the sodium butyrate effect on deacetylases. Since we have previously found that such a protein would also mediate the sodium butyrate effect on gene expression, we propose that a phosphatase activity constitutes an early and essential step in the sodium butyrate-triggered signalling pathway.

We here report EPR studies that provide evidence for radical intermediates generated from the glycyl radical of activated pyruvate formate-lyase (PFL) during the process of oxygen-dependent enzyme inactivation, radical quenching, and protein fragmentation. Upon exposure of active PFL to air, a long-lived radical intermediate was generated, which exhibits an EPR spectrum assigned to a sulfinyl radical (RSO*). The EPR spectrum of a sulfinyl radical was also generated from the activated C418A mutant of PFL, indicating that Cys 418 is not the site of sulfinyl radical formation. Exposure of the activated C419A mutant or C418AC419A double mutant to air on the other hand, resulted in a new EPR spectrum that we assign to the alpha-carbon peroxyl radical (ROO*) of the active-site glycine, G734. These findings suggest that C419 is the site of sulfinyl radical formation and that replacement of this cysteine with alanine results in the accumulation of the carbon peroxyl radical. The results also support the proposal that the peroxyl radical and the sulfinyl radical are intermediates in the oxygen-dependent inactivation and cleavage of the protein. Moreover, these observations are consistent with the hypothesis that C419 and G734 are in close proximity in the activated enzyme and may participate in a glycyl/thiyl radical equilibrium. A mechanism that accounts for the formation of the radical intermediates is proposed.

Impaired bone formation resulting from a decline of osteoblast activity has been implicated in the pathogenesis of diabetic osteopenia. We examined the effects of high glucose concentration alone, independent of insulin deficiency, on the growth of a human osteoblast-like cell line (MG-63). Sustained exposure to high glucose for 7 days inhibited cell growth in a concentration-dependent manner up to 49.5 mmol/L, as compared with cells cultured with a normal glucose concentration (5.5 mmol/L) or a high mannitol concentration (an iso-osmolar control). Glucose (49.5 mmol/L) attenuated the increment either in DNA content or in [3H]thymidine incorporation induced by insulin-like growth factor I (IGF-I). The IGF-I-induced increase of ornithine decarboxylase (ODC) activity, which plays an important role in cell growth, was also attenuated. The half-life of ODC protein was not shortened by the high glucose culture, but the intracellular content of putrescine (an end product of ODC) was significantly decreased. These changes did not occur in the high mannitol culture, strongly suggesting a specific effect of glucose. In summary, our observations suggest that a high glucose concentration significantly impairs the proliferative response of osteoblastic cells to IGF-I and that the defective cell function caused by sustained exposure to high glucose levels might contribute to impaired bone formation in patients with diabetic osteopenia.

Specific inhibitors of histone deacetylase, such as trichostatin A (TSA) and trapoxin (TPX), are potent inducers of HIV-1 transcription in latently infected T-cell lines. Activation of the integrated HIV-1 promoter is accompanied by the loss or rearrangement of a positioned nucleosome (nuc-1) near the viral RNA start site. Here we show that TSA strongly induces HIV-1 transcription on chromatin in vitro, concomitant with an enhancer factor-assisted increase in the level of acetylated histone H4. TSA treatment, however, did not detectably alter enhancer factor binding or the positioning of nuc-1 on the majority of the chromatin templates indicating that protein acetylation and chromatin remodeling may be limiting steps that occur only on transcriptionally competent templates, or that remodeling of nuc-1 requires additional factors. To assess the number of active chromatin templates in vitro, transcription was limited to a single round with low levels of the detergent Sarkosyl. Remarkably, HIV-1 transcription on chromatin was found to arise from a small number of active templates that can each support nearly 100 rounds of transcription, and TSA increased the number of active templates in each round. In contrast, transcription on naked DNA was limited to only a few rounds and was not responsive to TSA. We conclude that HIV-1 enhancer complexes greatly facilitate transcription reinitiation on chromatin in vitro, and act at a limiting step to promote the acetylation of histones or other transcription factors required for HIV-1 enhancer activity.

Lyso-platelet-activating factor (PAF) acetyltransferase is a critical regulatory step in PAF synthesis. PAF accumulates in the cornea in response to injury and is a potent inflammatory mediator that stimulates corneal cyclooxygenase but not lipoxygenase reactions. 12(S)-hydroxyeicosatetraenoic (HETE) acid, a major lipoxygenase product of mammalian corneas, is also generated during injury. In the bovine corneal epithelium, both PAF acetyltransferase and 12-lipoxygenase are microsomal enzymes. A potential interaction between these two lipid mediators was, therefore, examined. PAF acetyltransferase activity was assayed by determining radioactivity in the trichloroacetic acid-precipitated complex of [3H]PAF bound to albumin, formed after incubation of corneal epithelial microsomes with lyso-PAF and [3H]acetyl CoA. Lipoxygenase metabolism by bovine corneal epithelial microsomes was also studied. While 12(S)-HETE did not activate lyso-PAF acetyltransferase, PAF synthesis was decreased when microsomes were treated with lipoxygenase inhibitors. The IC50 values for nordihydroguaiaretic acid (NDGA), and baicalein were 75 and 105 microM, respectively. The IC50 value for CDC, a more potent inhibitor of platelet 12-lipoxygenase, was greater than 200 microM. The results indicate that concentrations suppressing lyso-PAF acetyltransferase activity exceed those required to inhibit lipoxygenases from bovine corneal epithelia. Similarly, concentrations of aspirin and indomethacin, cyclooxygenase inhibitors that decrease PAF formation, were greater than those reported to block prostaglandin generation. A number of other compounds, some structurally similar to the lipoxygenase inhibitors that suppress lyso-PAF acetyltransferase, and others unrelated chemically but known as anti-oxidant and cationic-chelators, also inhibited lyso-PAF acetyltransferase. This suggests that lyso-PAF acetyltransferase activity is inhibited by mechanisms independent of lipoxygenase and cyclooxygenase metabolites.

The spermine analog N1,N11-diethylnorspermine (DE-333, also known as DENSPM or BENSPM) is regarded as the most potent known inducer of the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), increasing activity by more than 200- to 1000-fold in certain cell types. The relative ability of a series of eight systematically modified DE-333 analogs to affect SSAT expression was examined in Malme-3M human melanoma cells, one of several cell lines known to be especially responsive to induction of this enzyme. In particular, we examined the relative contribution of induction of enzyme mRNA and prolongation of enzyme half-life to analog-mediated increases in enzyme activity. Induction of enzyme mRNA was most influenced by intra-amine carbon distances; relative effectiveness was found to be proportional to the number of three-carbon units. Stabilization of enzyme was most determined by the terminal N-alkyl substituent size; among methyl, ethyl and propyl groups, methyl was least effective. Thus, DE-333, which most potently induces SSAT mRNA and effectively stabilizes SSAT enzyme activity, produces the greatest increase in enzyme activity. Although other contributing mechanisms may be involved, the relative abilities of the various analogs to induce enzyme activity is at least partially attributable to their combined effects on enzyme mRNA and protein half-life. These data reveal the highly sensitive structure-activity relationships that underlie and control spermine analog induction of SSAT activity. Pending further definition of the relationship between SSAT induction and antitumor growth and toxicity in vivo, these relationships may be used to optimize therapeutic efficacy.

We earlier developed the Chinese hamster ovary UV5P3 cell line that expresses cytochrome P4501A2 and lacks nucleotide excision repair for studying metabolism and mutagenicity of heterocyclic amines. The Chinese hamster ovary UV5P3 cells are approximately 50-fold more sensitive to the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) than 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), another genotoxic compound found in cooked food, with respect to cytotoxicity and mutation induction at the adenine phosphoribosyltransferase (aprt) locus. To test the hypothesis that the important missing activity in our CHO system for IQ genotoxicity was acetyltransferase, we transfected the UV5P3 cells with cDNA plasmids of either the human NAT2 N-acetyltransferase gene or a bacterial O-acetyltransferase gene. Functionally transformed clones were determined by the differential cytotoxicity assay using IQ, and confirmed by measuring the enzyme activity with isoniazid as substrate. Two clones designated 5P3NAT2 and 5P3YG (expressing human and bacterial transferases, respectively) were characterized. Both cell lines were sensitive to killing by IQ at concentrations as low as 4 ng/ml. Based on the D37 value, the dose that reduced the survival to 37% relative to untreated controls, the acetyltransferase expressing lines showed approximately 1000-fold increase in sensitivity to the killing effect of IQ over the parental UV5P3 cell line. The same dramatic change in sensitivity was also seen in mutation response at the aprt locus and with chromosomal aberrations and sister chromatid exchanges. In contrast, these cell lines showed cytotoxicity to PhIP similar to that of the parental line UV5P3. These results suggest that PhIP does not require acetyltransferase for metabolic activation leading to genotoxicity in these cells. These new cell lines constitute a sensitive cell system for assessing genotoxicity of compounds requiring metabolic activation by both P450IA2 and acetyltransferase, as well as for studying the molecular processes by which DNA damage can lead to mutation and cancer.

Neuronal nuclear fraction N1 was isolated from cerebral cortices of 15-day-old rabbits, and nuclear subfractions prepared, in order to study the location of nuclear lyso platelet-activating factor (lyso-PAF) acetyltransferase and alkylglycerophosphate (AGP) acetyltransferase, and factors that affect the loss of these two nuclear activities. Subfractionation of prelabelled N1 indicated that the nuclear envelope had the highest percentage of the radioactive acetylated products alkylacetylglycerophosphate (AAGP) and PAF, and the distribution of these phospholipids reflected phospholipid distributions in the nuclear subfractions. The majority (95%) of radioactive AAGP and PAF was also recovered in Triton X-100 extracts of prelabelled nuclei, suggesting that these acetylated lipids are located in nuclear membranes rather than in the nuclear matrix/chromatin. Of the nuclear subfractions, the envelope had the highest AGP and lyso-PAF acetyltransferase specific activities which were close to corresponding values seen in the parent N1 fraction. Thus the nuclear AGP and lyso-PAF acetyltransferases were principally localized to the nuclear membranes. Differentials in activity loss were seen for the two acetyltransferase activities. In the nuclear envelope fractions, the lyso-PAF acetyltransferase was the more susceptible to oxidation reactions which could be reversed or blocked by the use of reducing agents. In preincubations, N1 showed greater losses in lyso-PAF acetyltransferase activity than in AGP acetyltransferase activity, losses which were not attributable to oxidation. Addition of cytosolic fraction S3 to preincubations promoted losses for each acetyltransferase in N1, and gave evidence for cytosolic and endogenous nuclear contributions to the activity loss. Addition of okadaic acid to the preincubations did not prevent the decline of either acetyltransferase in intact nuclei, but did diminish the loss of nuclear lyso-PAF acetyltransferase activity promoted by S3 addition, and also blocked the loss of this acetyltransferase seen in preincubations of isolated nuclear envelopes. This suggests that nuclear lyso-PAF acetyltransferase is susceptible to okadaic acid-sensitive nuclear and cytosolic protein phosphatase activities, while AGP acetyltransferase may lose activity by the action of other phosphatases or by other mechanisms within the nucleus.

Induction of the polyamine acetylating enzyme, spermidine/spermine N1-acetyltransferase (SSAT), is one of several biochemical effects associated with the antiproliferative action of polyamine analogs such as N1, N11 diethylnorspermine (DENSPM). Findings to date indicate that this complex and extremely potent gene response involves increased gene transcription, stabilization of mRNA, enhanced translation and protein stabilization. In this study, SSAT-directed antisense oligonucleotide analogs (AOs) were studied for their ability to prevent enzyme induction by DENSPM. Nine 18-mer fully phosphorothioate modified AOs targeting the start codon, exon 6, stop codon and polyadenylation regions of the human SSAT mRNA were synthesized and evaluated in MALME-3M human melanoma cells prior to and during a 6 hr treatment with 10 microM DENSPM. The most effective AOs were those targeting sequences in the stop codon region. Of these, AO-82 suppressed DENSPM induction of SSAT activity, enzyme protein and mRNA by 70-80%. The quantitative similarity of these effects suggests AO interference with mRNA stabilization, a property apparently mediated by sequences located in the stop codon region. Growth inhibition by DENSPM in the presence of the terminally phophorothioated analogs of AO-82 remained similar to that produced by DENSPM alone. While it is possible that SSAT induction may not be involved in analog-mediated antiproliferative activity, a more likely interpretation is that the approximately 50% suppression of the enzyme response achieved in growth studies is not sufficient to abrogate growth inhibition.

The effects of (Z)-11-[(3-dimethylamino)propylidene]-6,11-dihydrodibenz [b.e.]oxepin-2-acetic acid monohydrochloride (KW-4679), an orally active antiallergic drug, on the production of platelet-activating factor (PAF), leukotriene (LT) and thromboxane (TX) induced by Ca2+ ionophore A23187 were examined. KW-4679 at 10 microM reduced the amount of cell-associated PAF by 52.8% in human polymorphonuclear leukocytes (PMNs). KW-4679 (1-100 microM) also inhibited the release of both LTB4 and TXB2, a stable metabolite of TXA2, by human PMNs in a concentration-dependent manner, but did not influence the release of beta-glucuronidase. The 50% inhibitory concentration (IC50) values for LTB4 and TXB2 release were 5.9 and 6.0 microM, respectively. In guinea pig eosinophils, KW-4679 inhibited the release of peptide LTs at a concentration higher than 10 microM (IC50 = 66.9 microM). KW-4679 failed to inhibit PAF acetyltransferase, 5-lipoxygenase and TX synthase, but inhibited the arachidonic acid release by human PMNs in a concentration-dependent manner in a similar concentration as that inhibiting production or release of lipid mediators (IC50 = 19.5 microM). These results indicate that KW-4679 suppresses LTs and TX release and PAF formation by reducing arachidonic acid release from phospholipids, probably through interference with phospholipase A2. The inhibitory action of KW-4679 on PAF, LT and TX production is a beneficial effect of an antiallergic drug.

The kinetic constants of the aminoglycoside-modifying enzyme 6'-N-aminoglycoside acetyltransferase (AAC(6')IV) from the clinical strain Staphylococcus epidermidis RYC 13036 differed depending on whether tobramycin and amikacin (glycosamine group) or gentamicin and netilmicin (garosamine group) were used as substrates. Acetylation of the glucosamine antibiotics was highly susceptible to substrate inhibition which increased with pH whereas the garosamine group compounds showed limited substrate inhibition over a wide pH range. These differences in activity correlated with MIC values of S. epidermidis RYC 13036 for different aminoglycosides. Aminosugars moiety and pH markedly influenced the AAC(6')IV-aminoglycoside interactions.

Nitroscanate (NSC) was found to be a direct acting mutagen in the Ames Salmonella tester strains TA100 and TA98 and this activity increased further in the presence of rat liver S9 mix. It was inactive in TA98NR and TA100NR, and weakly active in TA98/1,8-DNP6. A substantial fall in drug induced mutagenicity by pentachlorophenol, an inhibitor of acetyltransferase, in TA98, TA100 and YG1024 suggests the initial bioconversion of a nitro group to hydroxylamine and its further activation to the ultimate N-acetoxyarylamine. The refrigerated DMSO solution of the drug in the plate incorporation assay and freshly prepared solutions using the pre-incubation procedure indicated a fall in mutagenicity owing to the conversion of NSC to N,N'-bis-4-(p-nitrophenoxy)phenyl thiourea (NFPT). The drastic reduction in mutagenicity in the presence of 4-amino-4'-nitrodiphenyl ether (ANDE) and 4-aminodiphenyl ether (ADE) was also attributed to the conversion of NSC to the corresponding thiourea, a non-mutagen. The negligible mutagenicity of ANDE and its absence in ADE and 4-isothiocyanate diphenyl ether (ITDE) suggests that the mutagenicity of NSC is due to the nitro group, and the -NCS function is responsible for enhanced mutagenicity over nor-isothiocyanate 4-nitrodiphenyl ether (NDE).

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) associate to form a large inner core with a protruding structure composed of three globular domains connected by mobile linker regions. This exterior region of E2 includes two lipoyl domains which engage not only in the intermediate reactions of the complex but also have integral roles in the kinase-phosphatase regulatory interconversion of the pyruvate dehydrogenase (E1) component. To facilitate understanding of these roles, lipoyl domain constructs of the E2 component of human PDC were expressed as glutathione S-transferase (GST)-linked fusion proteins from plasmid inserts prepared by polymerase chain reaction procedures. The NH2-terminal lipoyl domain, E2L1, and the interior lipoyl domain, E2L2, are connected by a 30-amino-acid hinge region, H1. Constructs designed and expressed were E2L1(1-98), E2L1.H1(1-128), E2L2(120-233), E2H1.L2(98-233), and E2L1.H1.L2(1-233), where numbers in parentheses give the amino acid sequence for the portions of the E2 component incorporated into a construct. The domains were expressed in Escherichia coli with and without lipoate supplementation. GST constructs were purified to homogeneity by affinity chromatography and selectively released by thrombin treatment. Sequencing of insert DNAs and NH2-terminal sequencing confirmed that domains were produced as designed. Measurement of masses by electrospray mass spectrometry indicated that constructs with lipoylated, nonlipoylated, and octanoylated forms were produced when expression was with E. coli grown without lipoate supplementation and that fully lipoylated forms were produced upon lipoate supplementation. The lipoylation status was confirmed, following delipoylation with Enterococcus faecalis lipoamidase, by the expected decrease in mass and by the observation in native gel electrophoresis of a shift to a slower mobility (possibly less compact) form. Constructs were used in E1-catalyzed reductive-acetylation reaction in proportion to their degree of lipoylation and were effective substrates in a NADH-dependent dihydrolipoyl dehydrogenase reduction reaction. Thus, we have produced lipoyl domain constructs that can be employed in sorting the specific roles of E2L1 and E2L2 in facilitating catalytic and regulatory processes.

1. Berenil, administered to rats in vivo, promoted a decrease in liver SAMDC activity, but an increase in ODC and SAT activity. 2. Its effect on ODC was completely prevented by cycloheximide, that on SAT only partially. 3. Berenil had no effect on ODC activity in adrenalectomized rats. Adrenergic antagonists counteracted the effect of Berenil on ODC activity. 4. Polyamine content was increased. The maximum modification was observed for putrescine and N1-acetylspermidine.

In in vitro systems, the spermine analogue, N1,N11-bis(ethyl)norspermine (BENSPM), suppresses the polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase (ornithine decarboxylase and S-adenosylmethionine decarboxylase, respectively), greatly induces the polyamine catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT), depletes polyamine pools, and inhibits cell growth. Against MALME-3 M human melanoma xenografts, BENSPM and related homologues demonstrate potent antitumor activity that has been found to correlate positively with their ability to induce SSAT activity in vitro. Herein, we further evaluate the antitumor activity of BENSPM and at the same time characterize the biochemical effects of BENSPM treatment on polyamine metabolism of selected normal and tumor tissues. At 40 mg/kg 3 times/day for 6 days i.p., BENSPM suppressed growth of MALME-3 M human melanoma xenografts during treatment and for 65 days afterwards. Similar antitumor activity was obtained with 120 mg/kg once daily for 6 days and 40 mg/kg once daily for 6 days, indicating that against this tumor model, the dosing schedule can be relaxed up to sixfold without compromising antitumor activity. When MALME-3 M tumor-bearing mice were retreated with BENSPM 2 weeks after the first treatment at 40 mg/kg 3 times/day for 6 days, initial tumor volumes of 85 mm3 were reduced to < 10 mm3. Analysis of melanoma, liver, and kidney tissues from mice treated with 40 mg/kg 3 times/day for 6 days revealed relatively similar accumulations of BENSPM in all tissues at levels greater than the original total content of polyamine pools. By 2 weeks following treatment, BENSPM pools in normal tissues were almost gone, whereas in tumor tissues significant amounts (40%) were still retained. The biosynthetic enzymes, ornithine decarboxylase and S-adenosylmethionine decarboxylase, gave no indication of enzyme suppression (or increase) by the analogue as typically occurs in vitro. By contrast, SSAT was induced from an average of < 50 pmol/min/mg in control tissues to 320 pmol/min/mg in liver, 1255 pmol/min/mg in kidney, and 13,710 pmol/min/mg in MALME-3M tumor. Two weeks later, SSAT activity was still 12 times higher in tumor than in kidney. Polyamine pools (putrescine, spermidine, and spermine) were reduced after treatment in all tissues and approached near-total depletion in the tumor. Good antitumor activity and even more potent induction of SSAT (i.e., 26,680 pmol/min/mg) was also observed in PANUT-3 human melanoma xenografts. Overall, the findings reveal meaningful antitumor activity by BENSPM against 2 human melanoma xenografts and provide in vivo evidence consistent with SSAT-induced polyamine depletion playing a determining role in at least the initial phase of the antitumor response.

The role of histone acetylation and DNA synthesis has been investigated extensively in the regenerating rat liver system in the presence and absence of the cyclophosphamide derivative mafosfamide. We demonstrate a mafosfamide-induced inhibition of maximum histone acetyltransferase activity followed by a second elevation of enzyme activity and an accompanying total suppression of DNA synthesis for 7-8 h. The maximum of histone acetyltransferase activity, in parallel with an elevated acetylation in vivo, the consecutive replacement of histone H1(0) amd initiation of replication occur sequentially in the presence and absence of mafosfamide, but with a temporary delay of 7-8 h. Our data indicate that modifications of histone acetyltransferase (EC 2.3.1.48) activity do not significantly influence the acetylation patterns of histones H3 and H4. The mafosfamide-induced change of histone acetyltransferase activity and acetylation in vivo, the shift of histone H1(0) exchange and the consecutive transition of initiation of replication suggest that these three events might be functionally related.

The activity of the platelet-activating factor (PAF)-synthesizing enzyme, 1-O-alkyl-sn-glycero-3-phosphocholine (lysoPAF):acetyl-CoA acetyltransferase (EC 2.3.1.67) in alveolar macrophage lysate was found to be elevated after warming the cells to 37 degrees C. Such an increase in enzyme activity was detectable only when intact cells were warmed. The stimulation was transient, reaching a peak at 2 min, and then gradually decreased to the control level. We could not find increased PAF formation in warmed cells which had increased acetyltransferase activity, even though substantial amounts of lysoPAF were shown to be present within cells. In contrast, considerable amounts of PAF were formed after treatment of the cells with exogenous lysoPAF. These results suggest that the activation of acetyltransferase is not sufficient to induce PAF formation and that the increased availability of substrates, especially lysoPAF, in the cells is indispensable for triggering PAF biosynthesis in this type of cells.