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This study aimed to treat diabetic cerebral ischemia-reperfusion injury (CI/RI) by affecting blood brain barrier (BBB) permeability and integrity. The CI/RI model in DM mice and a high glucose (HG) treated oxygen and glucose deprivation/reoxygenation (OGD/R) brain endothelial cell model were established for the study. Evans blue (EB) staining was used to evaluate the permeability of BBB in vivo. TTC staining was used to analyze cerebral infarction. The location and expression of tribbles homolog 3 (TRIB3) in endothelial cells were detected by immunofluorescence. Western blotting was used to detect the protein expressions of TRIB3, tight junction molecules, adhesion molecules, phosphorylated protein kinase B (p-AKT) and AKT. The levels of pro-inflammatory cytokines were detected by qRT-PCR. Trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran were used to measure vascular permeability in vitro. TRIB3 ubiquitination and acetylation levels were detected. Acetyltransferase bound to TRIB3 were identified by immunoprecipitation. TRIB3 was localized in cerebral endothelial cells and was highly expressed in diabetic CI/R mice. The BBB permeability in diabetic CI/R mice and HG-treated OGD/R cells was increased, while the junction integrity was decreased. Interference with TRIB3 in vitro reduces BBB permeability and increases junction integrity. In vivo interfering with TRIB3 reduced cerebral infarction volume, BBB permeability and inflammation levels, and upregulated p-AKT levels. The phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin reversed the effects of TRIB3-interfering plasmid. In vitro HG treatment induced TRIB3 acetylation through acetyltransferase p300, which in turn reduced ubiquitination and stabilized TRIB3. Interfering TRIB3 protects BBB by activating PI3K/AKT pathway and alleviates brain injury, which provides a new target for diabetic CI/RI., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Apoptosis of skin keratinocytes is closely associated with skin problems in humans and natural flavonoids have shown excellent biological activity. Hence, the study of flavonoids against human keratinocyte apoptosis has aroused the interest of numerous researchers. In this study, methyl thiazolyl tetrazolium (MTT) assay and Western blots were used to investigate the skin-protective effect of isoviolanthin, a di-C-glycoside derived from Dendrobium officinale, on hydrogen peroxide (H2O2)-triggered apoptosis of skin keratinocytes. Transcriptome sequencing (RNA-Seq) was used to detect the altered expression genes between the model and treatment group and qRT-PCR was used to verify the accuracy of transcriptome sequencing results. Finally, molecular docking was used to observe the binding ability of isoviolanthin to the selected differential genes screened by transcriptome sequencing. Our results found isoviolanthin could probably increase skin keratinocyte viability, by resisting against apoptosis of skin keratinocytes through downregulating the level of p53 for the first time. By comparing transcriptome differences between the model and drug administration groups, a total of 2953 differential expression genes (DEGs) were identified. Enrichment analysis showed that isoviolanthin may regulate these pathways, such as DNA replication, Mismatch repair, RNA polymerase, Fanconi anemia pathway, Cell cycle, p53 signaling pathway. Last, our results found isoviolanthin has a strong affinity for binding to KDM6B, CHAC2, ESCO2, and IPO4, which may be the potential target for treating skin injuries induced by reactive oxide species. The current study confirms isoviolanthin potential as a skin protectant. The findings may serve as a starting point for further research into the mechanism of isoviolanthin protection against skin damage caused by reactive oxide species (e.g., hydrogen peroxide)., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

N-acetyltransferase 10 (NAT10), a nuclear acetyltransferase and a member of the GNAT family, plays critical roles in RNA stability and translation processes as well as cell proliferation. Little is known about regulatory effects of NAT10 in lung epithelial cell proliferation. We firstly investigated NTA10 mRNA expression in alveolar epithelial types I and II, basal, ciliated, club, and goblet/mucous epithelia from heathy and patients with chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, lung adenocarcinoma, para-tumor tissue, and systemic sclerosis, respectively. We selected A549 cells for representative of alveolar epithelia or H1299 and H460 cells as airway epithelia with different genetic backgrounds and studied dynamic responses of NAT10-down-regulated epithelia to high temperature, lipopolysaccharide, cigarette smoking extract (CSE), drugs, radiation, and phosphoinositide 3-kinase (PI3K) inhibitors at various doses. We also compared transcriptomic profiles between alveolar and airway epithelia, between cells with or without NAT10 down-regulation, between early and late stages, and between challenges. The present study demonstrated that NAT10 expression increased in human lung epithelia and varied among epithelial types, challenges, and diseases. Knockdown of NAT10 altered epithelial mitochondrial functions, dynamic responses to LPS, CSE, or PI3K inhibitors, and transcriptomic phenomes. NAT10 regulates biological phenomes, and behaviors are more complex and are dependent upon multiple signal pathways. Thus, NAT10-associated signal pathways can be a new alternative for understanding the disease and developing new biomarkers and targets., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

We aimed to examine the effects of brain ischemia-reperfusion (IR) especially on serum parameters or liver enzymes, free radicals, cytokines, oxidatively damaged DNA, spermidine/spermine N-1-acetyltransferase (SSAT). The effects of addition of putrescine on IR will be evaluated in terms of inflammation and oxidant-antioxidant balance in liver.The study was conducted on 46 male Albino Wistar rats weighing 200-250 g. The rats were grouped into: 1-Sham group (n = 6). 2-IR group (n = 8): The carotid arteries were ligated for 30-min and reperfusion was achieved for 30-min under general anesthesia. 3-Ischemia + putrescine + reperfusion group (IPR) (n = 8): Unlike the IR group, a single dose of 250 μmol kg-1 putrescine was given by gavage at the beginning of reperfusion. In putrescine treatment groups in addition to the procedures performed in the IR group a total of 4 doses of 250 μmol kg-1 putrescine were given at 12-h intervals, with the first dose immediately after 30-min reperfusion (4-IR+putrescine group (IR+P1) (n = 8)); 3 h after the 30-min reperfusion (5-IR+putrescine group (IR+P2) (n = 8)); 6 h after the 30-min reperfusion (6-IR+putrescine group (IR+P3) (n = 8)). ALT, AST, ATP, NO, SSAT, 8-OHdG levels were analyzed in the serum, and liver samples. NF-κB and IL-6 levels were analyzed in the liver samples.Brain IR causes inflammatory, oxidative and DNA damage in the liver, and putrescine supplementation through gavage reduces liver damage by showing anti-inflammatory and antioxidant effects.

T-cell acute lymphoblastic leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which the histone deacetylase SIRT1 is overexpressed downstream of a NOTCH1-bound enhancer. SIRT1 loss impaired leukemia generation, whereas SIRT1 overexpression accelerated leukemia and conferred resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, pharmacologic or genetic inhibition of SIRT1 resulted in significant antileukemic effects. Global acetyl proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene-expression profiling revealed metabolic changes together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a nonacetylatable KAT7-mutant partly rescued SIRT1 loss-induced proliferation defects. Overall, our results uncover therapeutic targets in T-ALL and reveal a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer., Significance: We identify a T-ALL axis whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results reveal T-ALL therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance. This article is highlighted in the In This Issue feature, p. 1., (©2022 American Association for Cancer Research.)

Major depressive disorder (MDD) is one of the most debilitating and severe mental diseases globally. Increasing evidence has shown that epigenetics is critical for understanding brain function and brain disorders, including MDD. N-acetyltransferase 10 (NAT10), acting on histones, mRNA and other substrates, has been reported to be involved in epigenetic events, including histone acetylation and mRNA modifications. NAT10 is highly expressed in the brain. However, the potential effects of NAT10 on MDD are still unknown. Here, we exploited chronic mild stress (CMS) to induce anxiety- and depression-like behaviors in mice and found that the expression of NAT10 in the mouse hippocampus was upregulated after CMS treatment. Inhibition of NAT10 by pharmacological methods produced anxiolytic- and antidepressant-like effects. Neuron-specific overexpression of NAT10 in the hippocampus resulted in anxiety- and depression-like behaviors, accompanied by higher SIRT1 protein levels, and lower dendritic spine densities. Overall, it was found that elevation of NAT10 in hippocampal neurons is involved in the occurrence of anxiety- and depression-like behaviors, suggesting that NAT10 could be a potential new target for developing anxiolytics and antidepressants., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Previous studies reported that long noncoding RNA (lncRNA) ZFPM2-AS1 is upregulated in renal cell carcinoma (RCC). However, the biological role of lncRNA ZFPM2-AS1 in RCC has not been explored. In this study, we investigated the role of lncRNA ZFPM2-AS1 in the progression of RCC. Quantitative real-time polymerase chain reaction was used for gene expression analysis, and functional assays including Cell Counting Kit-8 assay, flow cytometry-based apoptosis assay and transwell migration assays were performed to examine the malignant phenotypes. The functional interaction between ZFPM2-AS1 or miR-130A-3P and their targets was detected by dual-luciferase reporter assay. We found that the expressions of ZFPM2-AS1 and ESCO2 were upregulated in RCC tissues and cells, whereas miR-130a-3p was downregulated. The expression level of ZFPM2-AS1 is significantly associated with advanced TNM, distant metastasis, lymphatic metastasis, and a poor overall survival in RCC patients. Silencing ZFPM2-AS1 in RCC cells suppressed cell proliferation, invasion, and migration, and induced cell apoptosis. ZFPM2-AS1 interacted with miR-130A-3P and negatively regulated its expression in RCC cells. We further showed that ESCO2 was a downstream target of miR-130a-3p. Both miR-130a-3p inhibitor and ESCO2 overexpression could rescue the inhibitory effects of ZFPM2-AS1 knockdown in RCC cells. Together, our study demonstrates that ZFPM2-AS1 plays an oncogenic role in RCC progression via the miR-130a-3p/ESCO2 axis., (© 2022 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia, Ltd on behalf of Kaohsiung Medical University.)

Injury from myocardial infarction (MI) and consequent post-MI remodeling is accompanied by massive loss of cardiomyocytes (CM), a cell type critical for contractile function that is for all practical purposes non-regenerable due to its profound state of proliferative senescence. Identification of factors that limit CM survival and/or constrain CM renewal provides potential therapeutic targets. Tip60, a pan-acetyltransferase encoded by the Kat5 gene, has been reported to activate apoptosis as well as multiple anti-proliferative pathways in non-cardiac cells; however, its role in CMs, wherein it is abundantly expressed, remains unknown. Here, using mice containing floxed Kat5 alleles and a tamoxifen-activated Myh6-MerCreMer recombinase transgene, we report that conditional depletion of Tip60 in CMs three days after MI induced by permanent coronary artery ligation greatly improves functional recovery for up to 28 days. This is accompanied by diminished scarring, activation of cell-cycle transit markers in CMs within the infarct border and remote zones, reduced expression of cell-cycle inhibitors pAtm and p27, and reduced apoptosis in the remote regions. These findings implicate Tip60 as a novel, multifactorial target for limiting the damaging effects of ischemic heart disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Extranodal natural killer/T cell lymphoma (ENKTL) is an aggressive malignant non- Hodgkin's lymphoma (NHL) with a poor prognosis. Therefore, novel therapeutic biomarkers and agents must be identified for the same. KAT5 inhibitor, NU 9056, is a small molecule that can inhibit cellular proliferation; however, its role in ENKTL has not been studied., Objective: The present study investigated the effect of NU 9056 in ENKTL cells and explored the possible molecular mechanism for its antitumour effect., Methods: The role of NU 9056 in ENKTL cells was investigated through the Cell Counting Kit-8 assay, flow cytometry, Western blot, and real-time quantitative polymerase chain reaction assay., Results: NU 9056 inhibited ENKTL cell proliferation and induced G2/M phase arrest. NU 9056 also induced apoptosis by upregulating DR4, DR5, and caspase 8 expressions. Additionally, NU 9056 increased the expression of Bax, Bid, and cytochrome C and decreased the expression of Bcl-2, Mcl-1, and XIAP. Furthermore, NU 9056 activated endoplasmic reticulum (ER) stress and inhibited the JAK2/STAT3 signalling pathway. The p38 mitogen-activated protein kinase (MAPK) signalling pathway was also activated by NU 9056, and the ERK signalling pathway was suppressed in natural killer/T cell lymphoma cells., Conclusion: NU 9056 inhibited cell proliferation, arrested cell cycle in the G2/M phase, and induced apoptosis through the stimulation of ER stress, thus inhibiting the JAK2/STAT3 signalling pathway and regulating MAPK pathways in ENKTL cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Long known for their role in histone acetylation, recent studies have demonstrated that lysine acetyltransferases also carry out distinct "orphan" functions. These activities impact a wide range of biological phenomena including metabolism, RNA modification, nuclear morphology, and mitochondrial function. Here, we review the discovery and characterization of orphan lysine acetyltransferase functions. In addition to highlighting the evidence and biological role for these functions in human disease, we discuss the part emerging chemical tools may play in investigating this versatile enzyme superfamily.

Although rhodopsin is essential for sensing light for vision, it also mediates light-induced apoptosis of photoreceptors in mouse. RPE65, which catalyzes isomerization of all-trans retinyl fatty acid esters to 11-cis-retinol (11cROL) in the visual cycle, controls the rhodopsin regeneration rate and photoreceptor susceptibility to light-induced degeneration. Mutations in RPE65 have been linked to blindness in affected children. Despite such importance, the mechanism that regulates RPE65 function remains unclear. Through unbiased expression screening of a bovine retinal pigment epithelium (RPE) cDNA library, we have identified elongation of very long-chain fatty acids-like 1 (ELOVL1) and fatty acid transport protein 4 (FATP4), which each have very long-chain fatty acid acyl-CoA synthetase (VLCFA-ACS) activity, as negative regulators of RPE65. We found that the VLCFA derivative lignoceroyl (C24:0)-CoA inhibited synthesis of 11cROL, whereas palmitoyl (C16:0)-CoA promoted synthesis of 11cROL. We further found that competition of FATP4 with RPE65 for the substrate of RPE65 was also involved in the mechanisms by which FATP4 inhibits synthesis of 11cROL. FATP4 was predominantly expressed in RPE, and the FATP4-deficient RPE showed significantly higher isomerase activity. Consistent with these results, the regeneration rate of 11-cis-retinaldehyde and the recovery rate for rod light sensitivity were faster in FATP4-deficient mice than wild-type mice. Moreover, FATP4-deficient mice displayed increased accumulation of the cytotoxic all-trans retinaldehyde and hypersusceptibility to light-induced photoreceptor degeneration. Our findings demonstrate that ELOVL1, FATP4, and their products comprise the regulatory elements of RPE65 and play important roles in protecting photoreceptors from degeneration induced by light damage.

The emergence of multidrug-resistant (MDR) tuberculosis (TB) highlights the urgent need to understand the mechanisms of resistance to the drugs used to treat this disease. The aminoglycosides kanamycin and amikacin are important bactericidal drugs used to treat MDR TB, and resistance to one or both of these drugs is a defining characteristic of extensively drug-resistant TB. We identified mutations in the -10 and -35 promoter region of the eis gene, which encodes a previously uncharacterized aminoglycoside acetyltransferase. These mutations led to a 20-180-fold increase in the amount of eis leaderless mRNA transcript, with a corresponding increase in protein expression. Importantly, these promoter mutations conferred resistance to kanamycin [5 microg/mL < minimum inhibitory concentration (MIC)

Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis. The specific cellular gene alterations responsible for hepatocarcinogenesis are not well known. Previous works showed that loss of TIP30, also called CC3, a putative tumor suppressor, increased the incidence of hepatocellular carcinoma in mice, and some clinical samples of human HCC tissues had aberrant expression of TIP30. Here, we report that the introduction of TIP30 by an adenovirus vector into HCC cell lines that had decreased expressions of TIP30 inhibited cell proliferation, decreased anchorage-dependent growth, suppressed invasion through the extracellular matrix, and inhibited tumorigenesis in nude mice. Moreover, exogenous expression of Tip30 sensitized HCC cells to cytotoxic drugs and to apoptosis induced by tumor necrosis factor-related ligands in vitro. Ectopic expression of TIP30 in HCC cells enhanced p53 expression and decreased Bcl-2/Bcl-xL expression. Treatment of nude mice bearing subcutaneously established HCC tumors with a combination of an adenovirus expressing TIP30 and the cytotoxic drug 5-fluorouracil completely suppressed tumor growth and prolonged survival. In conclusion, TIP30 may play an important role in the suppression of hepatocarcinogenesis by acting as a tumor suppressor. Overexpression of TIP30 might be a promising candidate as a treatment for HCC that would increase sensitivity to chemotherapeutic drugs.

Our earlier observations led to the identification of a microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase) catalyzing the transfer of acetyl groups from acetylated polyphenols to the receptor proteins. TAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (1). The specificities of the acetoxy group on the benzenoid ring and position of the pyran carbonyl group of the coumarin with respect to oxygen heteroatom for the catalytic activity of TAase were also reported earlier. In this communication, we have demonstrated that the acetoxy coumarins and acetoxy dihydrocoumarins having a methyl group instead of a phenyl ring at the C-4, when used as the substrates, resulted in enhancement of TAase activity, while the saturation of double bond at C-3 and C-4 position had no effect on TAase activity. A comparison of the optimized structures of 1 and 7,8-diacetoxy-4-phenylcoumarin (2) suggested that the observed influence may be due to out of plane configuration of the phenyl ring at C-4. Further, the TAase-catalyzed activation of NADPH cytochrome c reductase and inhibition of aflatoxin B1 (AFB1)-DNA binding by acetoxy 4-phenylcoumarins and dihydrocoumarins were significantly lower as compared to those caused by acetoxy 4-methylcoumarins.

Curcumin (Cur), a well-known dietary pigment derived from Curcuma longa, is a promising anticancer drug, but its in vivo target molecules remain to be clarified. Here we report that exposure of human hepatoma cells to Cur led to a significant decrease of histone acetylation. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are the enzymes controlling the state of histone acetylation in vivo. Cur treatment resulted in a comparable inhibition of histone acetylation in the absence or presence of trichostatin A (the specific HDAC inhibitor), and showed no effect on the in vitro activity of HDAC. In contrast, the domain negative of p300 (a most potent HAT protein) could block the inhibition of Cur on histone acetylation; and the Cur treatment significantly inhibited the HAT activity both in vivo and in vitro. Thus, it is HAT, but not HDAC that is involved in Cur-induced histone hypoacetylation. At the same time, exposure of cells to low or high concentrations of Cur diminished or enhanced the ROS generation, respectively. And the promotion of ROS was obviously involved in Cur-induced histone hypoacetylation, since Cur-caused histone acetylation and HAT activity decrease could be markedly diminished by the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) or their combination, but not by their heat-inactivated forms. The data presented here prove that HAT is one of the in vivo target molecules of Cur; through inhibiting its activity, Cur induces histone hypoacetylation in vivo, where the ROS generation plays an important role. Considering the critical roles of histone acetylation in eukaryotic gene transcription and the involvement of histone hypoacetylation in the lose of cell viability caused by high concentrations of Cur, these results open a new door for us to further understand the molecular mechanism involved in the in vivo function of Cur.

The Wilms' tumor gene-1 (WT1) encodes a zinc finger protein involved in gene regulation during kidney, gonad, and heart development. In addition to its promoter, a 258 bp intronic enhancer is required for tissue-specific expression of WT1 gene. p300 is a histone acetyltransferase (HAT) and exerts essential functions in gene regulation. Here, we show that p300 increased the expression of endogenous WT1 mRNA and promoted the activation of the WT1 promoter and intronic enhancer. The results also revealed that the adenovirus E1A repressed the p300 function, while the p300-binding defective E1A delta 2-36 did not, and p300 HAT activity was important for its function since p300 mutant with the HAT domain deleted partially abrogated its ability to activate the WT1 promoter and intronic enhancer. Furthermore, p300 and c-Myb synergistically activated the expression of WT1 gene. This study revealed that p300 and its HAT activity were involved in regulation of WT1 transcription.

Although inhibition of histone deacetylase has been demonstrated to induce apoptosis of various cancer cells, there is no report on its effect on mast cell demise to date. Here we studied whether a histone deacetylase inhibitor Trichostatin A (TSA) produces apoptosis in p815 mastocytoma cells. TSA prominently increased the amount of acetylated histones, H3, H4, H2A and H2B, in p815 mastocytoma cells. TSA reduced the viability of p815 mastocytoma cells, and many apoptotic manifestations such as generation of DNA fragmentation, activation of caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), and increase of DNA hypoploidy proved that the reduction of viability resulted from apoptosis. Whereas TSA treatment increased the expression level of Bad, it decreased the level of Bcl-2, Bcl-xL, and X-linked inhibitor of apoptosis protein. The reduction of mitochondrial membrane potential, the release of cytochrome c and Smac/DIABLO to cytosol, and mitochondrial localization of Bad were also shown. Taken together, TSA induces apoptosis on p815 mastocytoma cells in histone acetylation- and mitochondria-dependent fashion. Our data therefore provide the possibility that TSA could be considered as a novel therapeutic strategy for mastocytoma from its apoptosis-inducing activity.

Aims: The present study is designed to investigate the acetylator status in Saudi Arabs., Methods: Isoniazid (INH) acetylation phenotyping was studied in 136 Saudi Arabs in Riyadh, Saudi Arabia, using a single plasma sample taken 3 h post-INH oral dose of 200 mg. Metabolic ratio (MR) of plasma acetyl-INH (Ac-INH) to INH was used to determine the acetylation phenotype., Results: The MR had a bimodal distribution with an antimode of 1.0. The frequency distribution of slow acetylators (MR < 1.0) was 94.9% (n = 129). Using Hardy-Weinberg Law, the gene frequency (q) of the recessive allele determining slow acetylator phenotype was found to be 0.97., Conclusion: INH phenotyping suggests a high frequency of slow acetylators among Saudi Arabs. There was no association between the MR of plasma Ac-INH/INH and age or gender.

Although the proximal cytoplasmic signaling events that control the activation of the NF-kappaB transcription factor are understood in considerable detail, the subsequent intranuclear events that regulate the strength and duration of the NF-kappaB-mediated transcriptional response remain poorly defined. Recent studies have revealed that NF-kappaB is subject to reversible acetylation and that this posttranslational modification functions as an intranuclear molecular switch to control NF-kappaB action. In this review, we summarize this new and fascinating mechanism through which the pleiotropic effects of NF-kappaB are regulated within the cells. NF-kappaB is a heterodimer composed of p50 and RelA subunits. Both subunits are acetylated at multiple lysine residues with the p300/CBP acetyltransferases playing a major role in this process in vivo. Further, the acetylation of different lysines regulates different functions of NF-kappaB, including transcriptional activation, DNA binding affinity, IkappaBalpha assembly, and subcellular localization. Acetylated forms RelA are subject to deacetylation by histone deacetylase 3 (HDAC3). This selective action of HDAC3 promotes IkappaBalpha binding and rapid CRM1-dependent nuclear export of the deacetylated NF-kappaB complex, which terminates the NF-kappaB response and replenishes the cytoplasmic pool of latent NF-kappaB/IkappaBalpha complexes. This readies the cell for the next NF-kappaB-inducing stimulus. Thus, reversible acetylation of RelA serves as an important intranuclear regulatory mechanism that further provides for dynamic control of NF-kappaB action.

Affinity chromatography was used to identify cellular proteins that interact with the herpes simplex virus (HSV) tegument protein VP22. Among a small set of proteins that bind specifically to VP22, we identified TAF-I (template-activating factor I), a chromatin remodelling protein and close homologue of the histone chaperone protein NAP-1. TAF-I has been shown previously to promote more ordered transfer of histones to naked DNA through a direct interaction with histones. TAF-I, as a subunit of the INHAT (inhibitor of acetyltransferases) protein complex, also binds to histones and masks them from being substrates for the acetyltransferases p300 and PCAF. Using in vitro assays for TAF-I activity in chromatin assembly, we show that VP22 inhibits nucleosome deposition on DNA by binding to TAF-I. We also observed that VP22 binds non-specifically to DNA, an activity that is abolished by TAF-I. However, the presence of VP22 does not affect the property of INHAT in inhibiting the histone acetyltransferase activity of p300 or PCAF in vitro. We speculate that this interaction could be relevant to HSV DNA organization early in infection, for example, by interfering with nucleosomal deposition on the genome. Consistent with this possibility was the observation that overexpression of TAF-I in transfected cells interferes with the progression of HSV-1 infection.

Histone acetyltransferases (HATs) are a group of enzymes that play a significant role in the regulation of gene expression. These enzymes covalently modify the N-terminal lysine residues of histones by the addition of acetyl groups from acetyl-CoA. Dysfunction of these enzymes is often associated with the manifestation of several diseases, predominantly cancer. Here we report that anacardic acid from cashew nut shell liquid is a potent inhibitor of p300 and p300/CBP-associated factor histone acetyltranferase activities. Although it does not affect DNA transcription, HAT-dependent transcription from a chromatin template was strongly inhibited by anacardic acid. Furthermore, we describe the design and synthesis of an amide derivative N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide (CTPB) using anacardic acid as a synthon, which remarkably activates p300 HAT activity but not that of p300/CBP-associated factor. Although CTPB does not affect DNA transcription, it enhances the p300 HAT-dependent transcriptional activation from in vitro assembled chromatin template. However, it has no effect on histone deacetylase activity. These compounds would be useful as biological switching molecules for probing into the role of p300 in transcriptional studies and may also be useful as new chemical entities for the development of anticancer drugs.

The induction of the monocyte chemoattractant protein 1 gene (MCP-1) by TNF occurs through an NF-kappaB-dependent distal regulatory region and an Sp1-dependent proximal regulatory region that are separated by 2.2 kb of sequence. To investigate how these regions coordinate activation of MCP-1 in response to TNF, experiments were performed to examine the role of coactivators, changes in local chromatin structure, and the acetylation of histones at the MCP-1 regulatory regions. An E1a-sensitive coactivator was found to be required for expression. In vivo nuclease sensitivity assays identified changes in response to TNF at both the proximal and distal regions that were dependent on the p65 subunit of NF-kappaB and Sp1. Chromatin immunoprecipitations used to analyze factor assembly and histone acetylation at the distal and proximal regions showed that Sp1 binding to and histone acetylation of the proximal region was dependent on NF-kappaB p65. Conversely, Sp1 assembly at the proximal region was required for p65 binding to and acetylation of the distal region, suggesting communication between the two regions during gene activation. These data and the NF-kappaB p65-dependent histone acetylation of a middle region sequence suggest a potential order for the assembly, acetylation and accessibility of the MCP-1 regulatory regions in response to TNF.

The HIV-1 Tat protein is required for viral replication and is a potent stimulator of viral transcription. Although Tat has been extensively studied in various reductive paradigms, to date there is little information as to how this activator mediates transcription from natural nucleosomally packaged long terminal repeats. Here we show that CREB-binding protein (CBP)/p300 interacts with the HIV-1 Tat protein and serves as a coactivator of Tat-dependent HIV-1 gene expression on an integrated HIV-1 provirus. The site of acetylation of Tat was mapped to the double-lysine motif in a highly conserved region, (49)RKKRRQ(54), of the basic RNA-binding motif of Tat. Using HLM1 cells (HIV-1(+)/Tat(-)), which contain a single copy of full-length HIV-1 provirus with a triple termination codon at the first AUG of the Tat gene, we find that only wild type, and not K50A, K51A, or K50A/K51A alone or in combination of ectopic CBP/p300, is able to produce full-length infectious virions, as measured by p24 gag ELISAs. Tat binds CBP/p300 in the minimal histone acetyltransferase domain (1253-1710) and the binding is stable up to 0.85 M salt wash conditions. Interestingly, wild-type peptide 41-54, and not other Tat peptides, changes the conformation of the CBP/p300 such that it can acquire and bind better to basal factors such as TBP and TFIIB, indicating that Tat may influence the transcription machinery by helping CBP/p300 to recruit new partners into the transcription machinery. Finally, using biotinylated wild-type or acetylated peptides, we find that acetylation decreases Tat's ability to bind the TAR RNA element, as well as to bind basal factors such as TBP, CBP, Core-Pol II, or cyclin T. However, the acetylated Tat peptide is able to bind to core histones on a nucleosome assembled HIV-1 proviral DNA., (Copyright 2000 Academic Press.)

The enzymatic transfer of acetyl groups from acetylated xenobiotics to specific proteins is a relatively grey area in the evergreen field of biotransformation of foreign compounds. In this paper, we have documented evidence for the existence of a transacetylase in liver microsomes that catalyses the transfer of acetyl groups from 7,8-diacetoxy-4-methylcoumarin (DAMC) to glutathione S-transferase (GST), either purified or present in cytosol leading to the irreversible inhibition of GST. A simple procedure is described for the assay of transacetylase by preincubation of DAMC with liver microsomes and pure GST/liver cytosol, followed by the addition of 1-chloro-2,4-dinitrobenzene (CDNB) and reduced glutathione (GSH) in order to quantify GST activity by the conventional procedure. The extent of inhibition of GST by DAMC under the conditions of the assay is indicative of DAMC:protein transacetylase activity. Following the assay procedure described here, the transacetylase was shown to exhibit hyperbolic kinetics. The bimolecular nature of the transacetylase reaction was apparent by the demonstration of Km and vmax values. 7,8-Dihydroxy-4-methylcoumarin (DHMC), one of the products of transacetylase reaction was identified and quantified using the partially purified enzyme. The fact that p-hydroxymercuribenzoate (PHMB) and iodoacetamide abolished irreversible inhibition of GST upon the action of transacetylase on DAMC strongly characterized transacetylase as a protein containing thiol group at the active site. In addition, the relative specificities of acetoxy 4-methylcoumarins to transacetylase have been demonstrated.

In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TRE(pal)) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.

From a rare actinomycete strain #8 isolated from soil as arbekacin (ABK) resistant, we cloned a gene segment (0.9 kb) conferring multiple resistance to aminoglycoside (AG) antibiotics with 6'-NH2 including semisynthetic ones except ABK and neomycin (NM). Enzymatic modification using cell free extracts from Streptomyces lividans TK21/pANT-S2 carrying the cloned gene revealed that the gene coded for an AG 6'-acetyltransferase [AAC(6')] capable of acetylating all of the tested AGs with 6'-NH2 including semisynthetic ones and astromicin. The substrate specificity of the enzyme was thus similar to that of AAC(6')-Ie of Enterococcus faecalis. Antibiotic assay revealed a weak but clear antibiotic activity of 6'-N-acetylABK (8% of ABK activity) in contrast with substantial inactivation by the AAC(6') of the other AGs including amikacin and isepamicin. The NM acetylation by the AAC(6') also did not result in NM inactivation. It seems thus likely that AAC(6')-dependent resistance to ABK and NM, if it emerges, will remain at low level.

The mutagenic activity of 17 substituted (aryl)(2-nitrobenzo[b]thiophen-3yl)amines has been evaluated in the Ames test with different isogenic strains of Salmonella typhimurium, that varied in their expression of nitroreductase and O-acetyltransferase. Active derivatives induced frameshift mutations in TA98 strain, and differences in the chemical structure resulted in up to 15-fold changes in mutagenic activity. The non-mutagenic compounds are the unsubstituted parent compound and derivatives with para-chloro, para-fluoro, para-diethylamino, meta-bromo and para-dimethylamino groups. They do not show any activity even in strains with higher level of nitroreductase or O-acetyltransferase. The addition of S9 fraction decreases the mutagenic potential or gives comparable results to those obtained without metabolic activation. For electron-donating substituents, the meta-isomers display the greatest mutagenic potency, whereas the transfer of the group to the para-position leads to less active or unactive molecules. All active nitrobenzothiophenes are substrates for bacterial nitroreductase and O-acetyltransferase, as shown by the reduced mutagenicity in the deficient strains and increased mutagenicity in the corresponding overproducing bacteria. Previous reports have pointed out interest in nitrothiophene analogues with para-chloro and para-fluoro substituents as promising anti-inflammatory drugs. The present study further enhances the putative interest in these derivatives, based on absence of mutagenicity.

We constructed the co-expression vector, pFK4, in which two cDNAs encoding serine acetyltransferase (SATase) and beta-(pyrazol-1-yl)-L-alanine/L-cysteine synthase (beta-PA/CSase) from Citrullus vulgaris (watermelon) were over-expressed under the transcriptional control of T7 promoter in Escherichia coli. Accumulation of both SATase and beta-PA/CSase in soluble extracts of E. coli was confirmed by immunoblotting. The high enzymatic activities of SATase and L-cysteine synthase (CSase) were detected in cell-free extracts of E. coli carrying pFK4. The activities of the formation of beta-PA and L-mimosine, plant non-protein amino acids, from O-acetyl-L-serine (OAS) and the precursor heterocyclic compounds, pyrazole and 3,4-dihydroxypyridine, were also found in the extracts. beta-PA was also produced in vivo from L-serine and pyrazole as precursors by E. coli cells transformed with pFK4. beta-PA was accumulated mainly in the extra-cellular culture medium. The pronounced accumulation of L-cysteine and L-methionine was observed in the cells transformed with pFK4. Additionally, we also constructed vectors which carried chimeric genes encoding fusion proteins of SATase and beta-PA/CSase. However, the fusion proteins tended to form insoluble inclusion bodies and thus to exhibit only weak enzymatic activities. The successful results of pFK4 shows the way to create a new sequential biosynthetic pathway of plant specific amino acids in bacterial cells by means of recombinant DNA technology.

To determine the postantibiotic effect of aminoglycosides, two methods are currently being used to remove the test drug: repeated washing and dilution. An enzymatic inactivation method of removing gentamicin and tobramycin was developed and compared with the dilution method. This enzymatic method provides a rapid and simple alternative method of removing aminoglycosides which results in reliable postantibiotic-effect values.

Human peripheral blood monocytes secrete tumor necrosis factor (TNF) in response to stimulation with bacterial lipopolysaccharide (LPS). We have shown that isolated human monocytes pretreated with LPS for 24 h secrete lower levels of TNF on a second stimulation with LPS than monocytes that have been stimulated with a single dose of LPS either immediately after isolation or 24 h after isolation. The levels of TNF released by monocytes after the second stimulation with LPS are proportional to the LPS concentration over a range from 1 ng/mL to 10 micrograms/mL. Increasing concentrations of LPS used during the first 24-h stimulation induce greater suppression of TNF release after a second stimulation with LPS. After an initial stimulus of 10 micrograms/mL LPS, a second stimulation of monocytes even with 10 micrograms/mL LPS will result in TNF secretion similar to that of unstimulated cells. This in vitro tolerance apparently can be overcome by stimulating previously activated cells with phorbol myristate acetate. We have also shown that neither prostaglandin E2 nor dexamethasone added during the initial stimulation with LPS had an effect on the subsequent reduction in TNF release on a second stimulation of monocytes with LPS.

Taurolidine (Geistlich Pharm, AG, Wolhusen, Switzerland), a derivative of the amino acid taurine, is commonly used in some parts of the world as an adjunctive therapy for various infections. Its mechanism of action is thought to be related to its antimicrobial properties, including its ability to interfere with some of the biological activities of endotoxin (lipopolysaccharide, LPS). For example, taurolidine has been shown to protect animals against endotoxic shock and death. In this study we examined the ability of taurolidine to block LPS-induced tumor necrosis factor (TNF) and interleukin 1 (IL-1) synthesis in human peripheral blood mononuclear cells (PBMC) from 27 donors. We observed a dose-dependent reduction in the synthesis of these two cytokines when taurolidine was preincubated with LPS before being added to PBMC. This reduction was independent of the molar ratio of taurolidine to LPS but was related to the concentration of taurolidine present in the PBMC cultures. There was a 80 to 90% reduction in total IL-1 and TNF synthesis induced by LPS at concentrations of taurolidine of 40 to 100 micrograms/mL; the vehicle was without effect. Following a 30-min preincubation with PBMC, taurolidine could be washed from the cells and still suppress cytokine synthesis induced by LPS. Using release of lactic acid dehydrogenase, 100 micrograms/mL of taurolidine was not toxic for PBMC. Taurolidine also reduced IL-1 and TNF synthesis induced by the Staphylococcus aureus-derived toxic shock syndrome toxin-1 as well as that induced by nontoxic heat-killed Staphylococcus epidermidis organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of thiols on the activation of a pyrolysate-derived N-hydroxyarylamine, 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-hydroxy-Glu-P-1), was studied in vitro. In hepatic cytosol of rats, [3H]-N-hydroxy-Glu-P-1 bound covalently to calf thymus DNA in the presence of acetyl CoA or 3'-phosphoadenosine-5'-phosphosulfate (PAPS). The extent of the binding of N-hydroxy-Glu-P-1 in a PAPS-dependent system was decreased by the addition of 10 mM glutathione, N-acetyl-L-cysteine, 2-mercaptoethanol or dithiothreitol. However, acetyl CoA-dependent binding of N-hydroxy-Glu-P-1 was stimulated by the addition of 10 mM N-acetyl-L-cysteine (3 fold), L-cysteine (2 fold) or glutathione (1.2 fold), but not 10 mM 2-mercaptoethanol or L-methionine. After hydrolysis of the modified DNA, no difference was detected in the physicochemical properties of the nucleoside adduct formed in the acetyl CoA-supported system with and without thiols. These results indicate that thiols with a cysteine residue are able to affect the activation of carcinogenic heterocyclic arylamines selectively by the modulation of the acetyltransferase-mediated, but not the sulfotransferase-mediated, pathway.

RESIDUAL GENTAMICIN ON A MILLIPORE MEMBRANE WAS INACTIVATED BY MODIFICATION WITH TWO DIFFERENT ENZYMES: gentamicin adenylyltransferase and 3-N-acetyltransferase. Both modifications allowed the growth of susceptible strains of Escherichia coli and Bacillus subtilis.

The isolation and structure determination of 3-N-acetylribostamycin, a microbiologically inactive derivative, produced enzymatically from ribostamycin by Streptomyces ribosidificus is described. The location of the acetyl group was established by mass and NMR spectrometry of the new compound and its derivatives, and by optical rotation studies conducted on N-ethoxycarbonyl-2-deoxystreptamine. The latter compound was obtained by partial acid hydrolysis of tri-N-ethoxycarbonyl-N-acetylribostamycin.

A new enzymatic activation of mutagenic N-hydroxyarylamines is described. An acetyl-CoA dependent enzyme that can activate 2-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) to a reactive species capable of binding to nucleic acid was found in a cell-free extract of Salmonella typhimurium TA98 but not in that of TA98/1,8-DNP6, which shows low sensitivity to the mutagenic activity of N-OH-Glu-P-1. The enzyme was partially purified by streptomycin treatment, ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration chromatography of Sephadex G-150. Its molecular weight was estimated to be approximately 48,000. The directly mutagenic N-hydroxyarylamines, such as N-OH-Glu-P-1, 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (N-OH-Trp-P-2), and N-hydroxy-2-aminofluorene (N-OH-AF), were transformed to reactive derivatives by partially purified enzyme in the presence of acetyl-CoA. The Km value for acetyl-CoA was calculated to be 3.3 microM. No acetyl residue, however, was incorporated into nucleic acid adducts. The enzymatic product of N-OH-Glu-P-1 bound most efficiently to polyguanylic acid among four polynucleotides. The enzyme did not show the N,O-acetyltransfer activity of N-hydroxyacetylaminofluorene (N-OH-AAF). These results indicate that the enzymatic product of N-hydroxyarylamine is N-acetoxyarylamine, and that this enzyme can be called acetyl-CoA:N-hydroxyarylamine O-acetyltransferase. O-Acetyltransferase activity was inhibited by SH-blocking agents, several phenolic compounds, such as pentachlorophenol and 1-nitro-2-naphthol, and an antibiotic thiolactomycin. S. typhimurium mutation studies suggested that the O-acetyltransferase functions as an enzyme activating certain N-hydroxyarylamines within bacterial cells and is involved in the formation of mutants.

Streptomyces tenebrarius ISP 5477, which produces nebramycins, was highly resistant to the following aminoglycoside antibiotics: neamine, ribostamycin, butirosin A, neomycin B, paromomycin, kanamycin A, dibekacin, gentamicin C complex, lividomycin A, istamycin B and streptomycin. Polyphenylalanine synthesis on the ribosomes of this strain was highly resistant to neamine, ribostamycin, butirosin A, kanamycins A, B and C, dibekacin, gentamicin C complex and istamycin B, moderately resistant to lividomycin A and streptomycin, but sensitive to neomycin B and paromomycin. Moreover, cell free extract of the strain contained phosphotransferase and N-acetyltransferase. The former enzyme was confirmed to be an aminoglycoside 6-phosphotransferase which inactivated streptomycin; the latter inactivated kanamycins B and C, dibekacin, neamine, neomycin B, paromomycin, lividomycin A, butirosin A and ribostamycin, but did not inactivate kanamycin A, gentamicin C complex and sagamicin, suggesting an aminoglycoside 2'-acetyltransferase. These results indicated that the high resistance of S. tenebrarius ISP 5477 to a wide range of aminoglycoside antibiotics is due to ribosomal resistance and to the inactivating enzymes, aminoglycoside N-acetyltransferase(s) and aminoglycoside 6-phosphotransferase.

Stability of aminoglycoside phosphotransferases, adenylyltransferases and acetyltransferases isolated from various sources was studied. The enzymes were characterized by different substrate profiles. They were stored at a temperature of -10 degrees C in the form of frozen solutions or at a temperature of 4 degrees C in the lyophilized form. It was shown that lyophilization markedly increased the stability of the enzymes inactivating aminoglycoside antibiotics. Aminoglycoside phosphotransferases and adenylyltransferases with streptomycin as substrate were less stable than aminoglycoside phosphotransferases with neomycin as substrate. Aminoglycoside acetyltransferases from Streptomyces fradiae 918 producing neomycin were least stable among the enzymes studied. Lyophilized enzymes as a possible stabilizer of ATP added to the preparations had no significant effect on their stability during storage.