Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli.

Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.

Characterization of a Peptide Antibody Specific to the Adenylyl Cyclase-Associated Protein of Acanthamoeba castellanii.

Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.

Application of Histone Deacetylase Inhibitors MPK472 and KSK64 as a Potential Treatment Option for Acanthamoeba Keratitis.

Treatment of Acanthamoeba keratitis (AK) is difficult because Acanthamoeba cysts are resistant to drugs, and as such, successful treatment requires an effective approach that inhibits cyst formation. Histone deacetylase inhibitors (HDACis) are involved in cell proliferation, differentiation, and apoptotic cell death. In this study, the effects of HDACis such as MPK472 and KSK64 on Acanthamoeba castellanii trophozoites and cysts were observed. MPK472 and KSK64 showed at least 60% amoebicidal activity against Acanthamoeba trophozoites at a concentration of 10 µM upon 8 h of treatment. Neither of the two HDACis affected mature cysts, but significant amoebicidal activities (36.4 and 33.9%) were observed against encysting Acanthamoeba following treatment with 5 and 10 µM HDACis for 24 h. Light microscopy and transmission electron microscopy results confirmed that the encystation of Acanthamoeba was inhibited by the two HDACis. In addition to this, low cytopathic effects on human corneal epithelial (HCE) cells were observed following treatment with MPK472 and KSK64 for 24 h. Our results indicate that the HDACis MPK472 and KSK64 could be used as new candidates for the development of an optimal therapeutic option for AK.

Combination of tert-butyl hydroperoxide with vorinostat induces cell death of Acanthamoeba through cell cycle arrest.

Safety precautions prior to contact lens usage is essential for preventing Acanthamoeba keratitis. Contact lens disinfecting solutions containing 3% hydrogen peroxide (H2O2) are known to exert amoebicidal effect against Acanthamoeba. Yet, these solutions need to be neutralized to prevent ocular irritation, which consequently may result in incomplete disinfection. In this study, amoebicidal effect of tert-butyl hydroperoxide (tBHP) was investigated and its efficacy was compared to those of hydrogen peroxide (H2O2). H2O2 and tBHP showed dose dependent amoebicidal effect, however high concentration of these compounds demonstrated cytotoxicity in human corneal epithelial (HCE) cells. To reduce their cytotoxicity, the concentrations of both compounds were diluted to 50 µM and subsequently combined with 10 µM vorinostat to enhance amoebicidal effect. Addition of vorinostat induced high amoebicidal effect against Acanthamoeba trophozoites, even at low concentrations of H2O2 or tBHP. Cellular damage induced by combined treatment of H2O2 or tBHP with vorinostat in Acanthamoeba were determined by assessing cell cycle arrest and apoptosis via FACS analysis. While 50 µM H2O2 combined with 10 µM vorinostat showed 36.26% cytotoxicity on HCE cells during 24 h exposure, 50 µM tBHP with 10 µM vorinostat did not show cytotoxicity on HCE cells. These findings suggest that the application of tBHP and vorinostat for Acanthamoeba keratitis treatment and contact lens disinfection system is highly plausible.

Upregulation of C/EBPß and TSC2 by an HDAC inhibitor CG200745 protects heart from DOCA-induced hypertrophy.

Histone deacetylases (HDACs) are a vast family divided into four major classes: class I (1, 2, 3, and 8), class II (4, 5, 6, 7, 9 and 10), class III (sirtuin family) and class IV (HDAC11). HDAC inhibition attenuates cardiac hypertrophy through suppression of the mechanistic target of rapamycin complex1 (mTORC1) signaling. HDAC inhibitors upregulate the expression of tuberous sclerosis complex 2 (TSC2), an mTORC1 inhibitor. However, the molecular mechanism underlying HDAC inhibitor-mediated upregulation of TSC2 is unclear. We hypothesized that an HDAC inhibitor, CG200745 (CG), ameliorates cardiac hypertrophy through the inhibition of mTORC1 signaling by upregulating of the CCAAT/enhancer-binding protein-ß (C/EBP-ß)/TSC2 pathway. To establish a cardiac hypertrophy model, deoxycorticosterone acetate (DOCA, 40 mg/kg/wk) was subcutaneously injected for 4 weeks into Sprague-Dawley rats. All rats were unilaterally nephrectomized and had free access to drinking water containing 1% NaCl with or without CG of different concentrations. The expression level of TSC2 and C/EBP-ß was measured by quantitative real-time PCR (qRT-PCR) and western blot analysis. Acetylation of C/EBP-ß was analyzed by immunoprecipitation. The recruitment of C/EBP-ß and polymerase II (Pol II) on TSC2 promoter region was analyzed by chromatin immunoprecipitation (ChIP). CG treatment increased the expression of TSC2. In addition, CG treated rats showed an increased in the expression and acetylation of C/EBP-ß, owing to the increase in the recruitment of C/EBP-ß and Pol II at Tsc2 gene promoter. Thus, CG ameliorates cardiac hypertrophy through the inhibition of mTORC1 signaling via upregulation of the C/EBP-ß/TSC2 pathway in DOCA-induced hypertensive rats.

Histone deacetylase inhibition ameliorates hypertension and hyperglycemia in a model of Cushing's syndrome.

Cushing's syndrome (CS) caused by hypercortisolism is occasionally accompanied by metabolic disorders such as hypertension, diabetes mellitus (DM), dyslipidemia, and central obesity. Thus morbidity and mortality, observed in cardiovascular disease, are elevated in patients with CS. We hypothesized that HDAC inhibition (HDACi) decreased transcriptional activity of glucocorticoid receptor (GR), which ameliorates hypertension and hyperglycemia in patients with CS. To establish an animal model of hypercortisolism, Sprague-Dawley rats were infused with adrenocorticotropic hormone (ACTH, 40 ng/day) or dexamethasone (Dex, 10 µg/day) via osmotic minipumps for 4 wk. Expression of GR target genes was determined by quantitative real-time PCR (qRT-PCR). GR enrichment on specific loci, and across the whole genome, was analyzed by chromatin immunoprecipitation (ChIP) and ChIPseq, respectively. HDACi decreased blood pressure and expression of ion regulators in the kidneys of ACTH-infused rats. Additionally, HDACi reduced deposition of polysaccharide, fasting blood glucose level, glucose intolerance, and expression of gluconeogenesis genes in the livers and kidneys of ACTH- and Dex-infused rats. Among class I HDACs, HDAC1 and HDAC3 interacted with GR. HDAC1 knockdown resulted in increased level of acetylation and decreased transcriptional activity of GR. GR recruitment on the promoters of 2,754 genes, which include ion transporters, channels, and gluconeogenic genes, was significantly decreased by MS-275, a class I HDAC inhibitor. These results indicate that HDACi ameliorates hypertension and hyperglycemia in a model of CS by decreasing the transcriptional activity of GR via elevating its level of acetylation.

Repression of Transcriptional Activity of Forkhead Box O1 by Histone Deacetylase Inhibitors Ameliorates Hyperglycemia in Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is a chronic disease manifested by hyperglycemia. It is essential to effectively control hyperglycemia to prevent complications of T2DM. Here, we hypothesize that repression of transcriptional activity of forkhead box O1 (FoxO1) via histone deacetylase inhibitors (HDACi) ameliorates hyperglycemia in T2DM rats. METHODS: Male Long-Evans Tokushima Otsuka (LETO) and Otsuka Long-Evans Tokushima Fatty (OLETF) rats aged 14 weeks were administered sodium valproate (VPA, 0.71% w/v) dissolved in water for 20 weeks. Electrophoretic mobility shift assay (EMSA) and luciferase assay were performed for elucidation of transcriptional regulation through acetylation of FoxO1 by HDACi. RESULTS: VPA attenuated blood glucose levels in accordance with a decrease in the expression of gluconeogenic genes in hyperglycemic OLETF rats. It has been shown that HDAC class I-specific and HDAC class IIa-specific inhibitors, as well as pan-HDAC inhibitors decrease FoxO1 enrichment at the cis-element of target gene promoters. Mutations in FoxO1 prevent its acetylation, thereby increasing its transcriptional activity. HDAC3 and HDAC4 interact with FoxO1, and knockdown of HDAC3, HDAC4, or their combination increases FoxO1 acetylation, thereby decreasing the expression of gluconeogenic genes. CONCLUSIONS: These results indicate that HDACi attenuates the transcriptional activity of FoxO1 by impeding deacetylation, thereby ameliorating hyperglycemia in T2DM rats.

Histone deacetylase inhibition attenuates hepatic steatosis in rats with experimental Cushing's syndrome.

Cushing's syndrome (CS) is a collection of symptoms caused by prolonged exposure to excess cortisol. Chronically elevated glucocorticoid (GC) levels contribute to hepatic steatosis. We hypothesized that histone deacetylase inhibitors (HDACi) could attenuate hepatic steatosis through glucocorticoid receptor (GR) acetylation in experimental CS. To induce CS, we administered adrenocorticotropic hormone (ACTH; 40 ng/kg/day) to Sprague-Dawley rats by subcutaneous infusion with osmotic mini-pumps. We administered the HDACi, sodium valproate (VPA; 0.71% w/v), in the drinking water. Treatment with the HDACi decreased steatosis and the expression of lipogenic genes in the livers of CS rats. The enrichment of GR at the promoters of the lipogenic genes, such as acetyl-CoA carboxylase (Acc), fatty acid synthase (Fasn), and sterol regulatory element binding protein 1c (Srebp1c), was markedly decreased by VPA. Pan-HDACi and an HDAC class I-specific inhibitor, but not an HDAC class II a-specific inhibitor, attenuated dexamethasone (DEX)-induced lipogenesis in HepG2 cells. The transcriptional activity of Fasn was decreased by pretreatment with VPA. In addition, pretreatment with VPA decreased DEX-induced binding of GR to the glucocorticoid response element (GRE). Treatment with VPA increased the acetylation of GR in ACTH-infused rats and DEX-induced HepG2 cells. Taken together, these results indicate that HDAC inhibition attenuates hepatic steatosis hrough GR acetylation in experimental CS.

Forkhead box O3 plays a role in skeletal muscle atrophy through expression of E3 ubiquitin ligases MuRF-1 and atrogin-1 in Cushing's syndrome.

Cushing's syndrome is caused by overproduction of the adrenocorticotropic hormone (ACTH), which stimulates the adrenal grand to make cortisol. Skeletal muscle wasting occurs in pathophysiological response to Cushing's syndrome. The forkhead box (FOX) protein family has been implicated as a key regulator of muscle loss under conditions such as diabetes and sepsis. However, the mechanistic role of the FOXO family in ACTH-induced muscle atrophy is not understood. We hypothesized that FOXO3a plays a role in muscle atrophy through expression of the E3 ubiquitin ligases, muscle RING finger protein-1 (MuRF-1), and atrogin-1 in Cushing's syndrome. For establishment of a Cushing's syndrome animal model, Sprague-Dawley rats were implanted with osmotic minipumps containing ACTH (40 ng·kg-1·day-1). ACTH infusion significantly reduced muscle weight. In ACTH-infused rats, MuRF-1, atrogin-1, and FOXO3a were upregulated and the FOXO3a promoter was targeted by the glucocorticoid receptor (GR). Transcriptional activity and expression of FOXO3a were significantly decreased by the GR antagonist RU486. Treatment with RU486 reduced MuRF-1 and atrogin-1 expression in accordance with reduced enrichment of FOXO3a and Pol II on the promoters. Knockdown of FOXO3a prevented dexamethasone-induced MuRF-1 and atrogin-1 expression. These results indicate that FOXO3a plays a role in muscle atrophy through expression of MuRF-1 and atrogin-1 in Cushing's syndrome.

Identification and Characterization of Protein Arginine Methyltransferase 1 in Acanthamoeba castellanii.

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.

DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation.

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.

Histone deacetylase inhibition, but not a mineralocorticoid receptor antagonist spironolactone, attenuates atypical transcription by an activating mutant MR (MRS 810L ).

A mutation in the mineralocorticoid receptor (MRS 810L ) leads to early-onset hypertension, which is markedly exacerbated during pregnancy. The mutation causes progesterone and even the MR antagonist spironolactone to become potent agonists. Thus, it is hard to control hypertension in patients harbouring this mutation. We hypothesized that histone deacetylase inhibition (HDACi), but not the MR antagonist spironolactone, attenuates atypical transcriptional activity of activating mutant MR (MRS 810L ). We established HEK293T cells overexpressing wild-type MR (MRWT ) or MRS 810L and determined their transcriptional activities by luciferase assay. Expression of MR target genes was measured by quantitative real-time PCR (qRT-PCR). Treatment with aldosterone increased the expression of MR target genes as well as the transcriptional activities in HEK293T cells transfected either with MRWT or MRS 810L . Treatment with either spironolactone or progesterone also increased the expression of MR target genes as well as transcriptional activity, but only in HEK293T cells transfected with MRS 810L . Spironolactone abolished the promoter activity stimulated by aldosterone in HEK293T cells transfected with MRWT . Treatment with HDAC inhibitors attenuated the transcriptional activity as well as the expression of MR target genes induced by aldosterone, spironolactone, or progesterone whether HEK293T cells were transfected with either MRWT or MRS 810L . These results indicate that HDACi, but not an MR antagonist spironolactone, attenuates atypical transcriptional activity of an activating mutant MR (MRS 810L ).

Histone deacetylase inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats used in this study including those in the sham group had been unilaterally nephrectomized and allowed free access to drinking water containing 1% NaCl. Systolic blood pressure was measured by the tail-cuff method. Blood chemistry including sodium, potassium, glucose, triglyceride, and cholesterol levels was analyzed. Sections of the heart were visualized after trichrome and hematoxylin and eosin stain. The expression of hypertrophic genes such as atrial natriuretic peptide A (Nppa) and atrial natriuretic peptide B (Nppb) in addition to fibrotic genes such as Collagen-1, Collagen-3, connective tissue growth factor (Ctgf), and Fibronectin were measured by quantitative real-time PCR (qRT-PCR). Injection of DOCA increased systolic blood pressure, heart weight, and cardiac fibrosis, which was attenuated by CG200745. Neither DOCA nor CG200745 affected body weight, vascular contraction and relaxation responses, and blood chemistry. Injection of DOCA increased expression of both hypertrophic and fibrotic genes, which was abrogated by CG200745. These results indicate that CG200745 attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

mGluR5 Upregulation increases excitability of hypothalamic presympathetic neurons through NMDA receptor trafficking in spontaneously hypertensive rats.

The hypothalamic paraventricular nucleus (PVN) is critically involved in elevated sympathetic output and the development of hypertension. However, changes in group I metabotropic glutamate receptors (mGluR1 and mGluR5) and their relevance to the hyperactivity of PVN presympathetic neurons in hypertension remain unclear. Here, we found that selectively blocking mGluR5 significantly reduced the basal firing activity of spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs), but not in normotensive Wistar-Kyoto (WKY) rats. However, blocking mGluR1 had no effect on the firing activity of PVN neurons in either group. The mRNA and protein levels of mGluR5 in the PVN and rostral ventrolateral medulla were significantly higher in SHRs than in WKY rats. The group I mGluR selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) similarly increased the firing activity of PVN neurons in WKY rats and SHRs. In addition, blocking NMDA receptors (NMDARs) through bath application or intracellular dialysis not only decreased the basal firing in SHRs, but also eliminated DHPG-induced excitation of spinally projecting PVN neurons. DHPG significantly increased the amplitude of NMDAR currents without changing their decay kinetics. Interestingly, DHPG still increased the amplitude of NMDAR currents and caused reappearance of functional NMDAR channels after initially blocking NMDARs. In addition, protein kinase C (PKC) inhibition or intracellular dialysis with synaptosomal-associated protein of 25 kDa (SNAP-25)-blocking peptide abolished DHPG-induced increases in NMDAR currents of PVN neurons in SHRs. Our findings suggest that mGluR5 in the PVN is upregulated in hypertension and contributes to the hyperactivity of PVN presympathetic neurons through PKC- and SNAP-25-mediated surface expression of NMDARs.

Histone deacetylase inhibition attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor in spontaneously hypertensive rats.

Inhibition of histone deacetylases (HDACs) by valproic acid (VPA) attenuates inflammatory, hypertrophic, and fibrotic responses in the hearts of spontaneously hypertensive rats (SHRs); however, the molecular mechanism is still unclear. We hypothesized that HDAC inhibition (HDACi) attenuates cardiac hypertrophy and fibrosis through acetylation of mineralocorticoid receptor (MR) in SHRs. Seven-week-old SHRs and Wistar-Kyoto rats were treated with an HDAC class I inhibitor (0.71% w/v in drinking water; VPA) for 11 weeks. Sections of heart were visualized after trichrome stain as well as H&E stain. Histone modifications, such as acetylation (H3Ac [acetylated histone 3]) and fourth lysine trimethylation (H3K4me3) of histone 3, and recruitment of MR and RNA polymerase II (Pol II) into promoters of target genes were measured by quantitative real-time polymerase chain reaction after chromatin immunoprecipitation assay. MR acetylation was determined by Western blot with anti-acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Treatment with VPA attenuated cardiac hypertrophy and fibrosis. Although treatment with VPA increased H3Ac and H3K4me3 on promoter regions of MR target genes, expression of MR target genes as well as recruitment of MR and Pol II on promoters of target genes were decreased. Although HDACi did not affect MR expression, it increased MR acetylation. These results indicate that HDACi attenuates cardiac hypertrophy and fibrosis through acetylation of MR in spontaneously hypertensive rats.

Histone deacetylase inhibition attenuates transcriptional activity of mineralocorticoid receptor through its acetylation and prevents development of hypertension.

RATIONALE: Inhibition of histone deacetylases (HDACs) results in attenuated development of hypertension in deoxycorticosterone acetate-induced hypertensive rats and spontaneously hypertensive rats. However, the molecular mechanism remains elusive. OBJECTIVE: We hypothesized that HDAC inhibition attenuates transcriptional activity of mineralocorticoid receptor (MR) through its acetylation and prevents development of hypertension in deoxycorticosterone acetate-induced hypertensive rats. METHODS AND RESULTS: Expression of MR target genes was measured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II on promoters of target genes was analyzed by chromatin immunoprecipitation assay. Live cell imaging was performed for visualization of nuclear translocation of MR. MR acetylation was determined by Western blot with anti-acetyl-lysine antibody after immunoprecipitation with anti-MR antibody. Transcriptional activity of MR was determined by luciferase assay. For establishment of a hyperaldosteronism animal, Sprague-Dawley rats underwent uninephrectomy and received subcutaneous injection of 40 mg/kg per week of deoxycorticosterone acetate and drinking water containing 1% NaCl. Treatment with a HDAC class I inhibitor resulted in reduced expression of MR target genes in accordance with reduced recruitment of MR and RNA polymerase II on promoters of target genes. HDAC inhibition promoted MR acetylation, leading to decreased transcriptional activity of MR. Knockdown or inhibition of HDAC3 resulted in reduced expression of MR target genes induced by mineralocorticoids. CONCLUSIONS: These results indicate that HDAC inhibition attenuates transcriptional activity of MR through its acetylation and prevents development of hypertension in deoxycorticosterone acetate-induced hypertensive rats.

Lysine acetyltransferases cyclic adenosine monophosphate response element-binding binding protein and acetyltransferase p300 attenuate transcriptional activity of the mineralocorticoid receptor through its acetylation.

Acetylation of the mineralocorticoid receptor (MR) by inhibition of lysine deacetylases attenuates MR's transcriptional activity. However, the specific lysine acetyltransferases that are responsible for acetylation of the MR remain unknown. We hypothesized that the acetyltransferases cyclic adenosine monophosphate response element-binding binding protein (CBP) and acetyltransferase p300 (p300) attenuate transcriptional activity of the MR through its acetylation. Expression of MR target genes was measured by quantitative real-time polymerase chain reaction. Recruitment of MR and RNA polymerase II (Pol II) on promoters of target genes was analysed by chromatin immunoprecipitation. Acetylation of the MR was determined by western blot with an anti-acetyl-lysine antibody after immunoprecipitation with an anti-MR antibody. In human embryonic kidney (HEK) 293 cells, overexpression of CBP or p300, but not p300/CBP-associated factor, increased MR acetylation and decreased expression of MR target genes. The downregulation of target genes coincided with a decrease in the recruitment of MR and Pol II to specific hormone response elements. These results demonstrate that overexpression of CBP or p300 attenuates the transcriptional activity of the MR through its acetylation in HEK 293 cells. Our data provide strong evidence identifying CBP and p300 as lysine acetyltransferases responsible for the regulation of MR that may provide new therapeutic targets for the treatment of hypertension.

Protein kinase CK2 contributes to diminished small conductance Ca2+-activated K+ channel activity of hypothalamic pre-sympathetic neurons in hypertension.

Small conductance calcium-activated K(+) (SK) channels regulate neuronal excitability. However, little is known about changes in SK channel activity of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) in essential hypertension. SK channels, calmodulin, and casein kinase II (CK2) form a molecular complex. Because CK2 is up-regulated in the PVN in spontaneously hypertensive rats (SHRs), we hypothesized that CK2 increases calmodulin phosphorylation and contributes to diminished SK channel activity in PVN pre-sympathetic neurons in SHRs. Perforated whole-cell recordings were performed on retrogradely labeled spinally projecting PVN neurons in Wistar-Kyoto (WKY) rats and SHRs. Blocking SK channels with apamin significantly increased the firing rate of PVN neurons in WKY rats but not in SHRs. CK2 inhibition restored the stimulatory effect of apamin on the firing activity of PVN neurons in SHRs. Furthermore, apamin-sensitive SK currents and depolarization-induced medium after-hyperpolarization potentials of PVN neurons were significantly larger in WKY rats than in SHRs. CK2 inhibition significantly increased the SK channel current and medium after-depolarization potential of PVN neurons in SHRs. In addition, CK2-mediated calmodulin phosphorylation level in the PVN was significantly higher in SHRs than in WKY rats. Although SK3 was detected in the PVN, its expression level did not differ significantly between SHRs and WKY rats. Our findings suggest that CK2-mediated calmodulin phosphorylation is increased and contributes to diminished SK channel function of PVN pre-sympathetic neurons in SHRs. This information advances our understanding of the mechanisms underlying hyperactivity of PVN pre-sympathetic neurons and increased sympathetic vasomotor tone in hypertension. Small conductance calcium-activated K(+) (SK) channels, calmodulin, and protein kinase CK2 form a molecular complex and regulate neuronal excitability. Our study suggests that augmented CK2 activity in hypertension can increase calmodulin (CaM) phosphorylation, which leads to diminished SK channel function in pre-sympathetic neurons. Diminished SK channel activity plays a role in hyperactivity of pre-sympathetic neurons in the hypothalamus in hypertension.

Detection of Acanthamoeba spp. using carboxylesterase antibody and its usage for diagnosing Acanthamoeba-keratitis.

Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.

Antiproliferation and Antiencystation Effect of Class II Histone Deacetylase Inhibitors on Acanthamoeba castellanii.

Acanthamoeba is a ubiquitous and free-living protozoan pathogen responsible for causing Acanthamoeba keratitis (AK), a severe corneal infection inflicting immense pain that can result in permanent blindness. A drug-based treatment of AK has remained arduous because Acanthamoeba trophozoites undergo encystment to become highly drug-resistant cysts upon exposure to harsh environmental conditions such as amoebicidal agents (e.g., polyhexanide, chloroquine, and chlorohexidine). As such, drugs that block the Acanthamoeba encystation process could result in a successful AK treatment. Histone deacetylase inhibitors (HDACi) have recently emerged as novel therapeutic options for treating various protozoan and parasitic diseases. Here, we investigated whether novel HDACi suppress the proliferation and encystation of Acanthamoeba. Synthetic class II HDACi FFK29 (IIa selective) and MPK576 (IIb selective) dose-dependently decreased the viability of Acanthamoeba trophozoites. While these HDACi demonstrated a negligible effect on the viability of mature cysts, Acanthamoeba encystation was significantly inhibited by these HDACi. Apoptosis was slightly increased in trophozoites after a treatment with these HDACi, whereas cysts were unaffected by the HDACi exposure. The viability of human corneal cells was not affected by HDACi concentrations up to 10 µmol/L. In conclusion, these synthetic HDACi demonstrated potent amoebicidal effects and inhibited the growth and encystation of Acanthamoeba, thus highlighting their enormous potential for further development.