OBJECTIVESTo study the effects of trichostatin A (TSA) on protein-protein interaction between HBx and histone deacetylase protein 1 (HDAC1).

METHODSBoth HBx and HDAC1 expressing vectors were constructed by the method of routine molecular cloning. The expression of HBx and HDAC1 were observed by Western blot assay. The protein-protein interaction was tested between HBx and HDAC1 by GST pull-down in vitro as well as co-immunoprecipitation in vivo.

RESULTSBoth HBx and HDAC1 expressing vectors were successfully constructed. Protein-protein interaction between HBx and HDAC1 existed both in vitro and in vivo. TSA, an inhibitor of HDAC1, had no effect on the interaction between HBx and HDAC1.

CONCLUSIONSHBx interacts with HDAC1 in vivo and in vitro in a non- TSA dependent way.

Histone Deacetylase 1 ; metabolism ; Humans ; Hydroxamic Acids ; metabolism ; Immunoprecipitation ; Plasmids ; Protein Interaction Mapping ; Trans-Activators ; metabolism

OBJECTIVE: To explore the analgesic mechanism of intrathecal trichostatin A (TSA) injection in a rat model of neuropathic pain induced by chronic constrictive injury (CCI). METHODS: Male SD rats were randomized into sham operation+ DMSO group (group S), CCI +DMSO group (group C), CCI +10 μg TSA group (group T), and in the latter two groups, rat models of neuropathic pain were established induced by CCI. The rats were given intrathecal injections of 10 μL 5% DMSO or 10 μg TSA (in 5% DMSO) once a day on days 7 to 9 after CCI or sham operation. The rats were euthanized after behavioral tests on day 10, and the lumbar segment of the spinal cord was sampled to determine the expression of histone deacetylase 4 (HDAC4) protein and mRNA and detect the differentially expressed miRNAs using a miRNA chip. MiR-190b-5p and miR-142-3p were selected for validation of the results using RT-qPCR. RESULTS: Compared with those in group S, the rats in group C showed significantly decreased paw withdrawal mechanical threshold (PWMT) from day 3 to day 10 after CCI ( < 0.05); intrathecal injection of TSA significantly reversed the reduction of PWMT following CCI ( < 0.05). Positive HDAC4 expression was detected mainly in the cytoplasm of the neurons in the gray matter of the spinal cord, and was obviously up-regulated after CCI ( < 0.05). Intrathecal injection of TSA significantly suppressed CCI-induced up-regulation of HDAC4 at 10 days after the operation ( < 0.05). Compared with the miRNA profile in group S, miRNA profiling identified 83 differentially expressed miRNAs in group C (fold change ≥2 or ≤0.5, < 0.05); TSA treatment reversed the expressions of 58 of the differentially expressed miRNAs following CCI, including 41 miRNAs that were decreased after CCI but up-regulated following TSA treatment. The results of real-time PCR validated the changes in the expressions of miR-190b-5p and miR-142-3p. CONCLUSIONS TSA suppresses CCI-induced up-regulation of HDAC4 and reverses differential expressions of miRNAs in the spinal cord of rats, which may contribute to the analgesic effect of TSA on neuropathic pain.
Animals ; Histone Deacetylases ; Hydroxamic Acids ; Male ; MicroRNAs ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; Up-Regulation

Objective: To explore effects of histone deacetylase inhibitor Belinostat on the immunologic function of dendritic cells (DC) and its possible mechanism. Methods: Cultured mouse bone marrow-derived DC from C57BL/6 mouse in vitro. The experiments were divided into 0, 50, 100 nmol/L Belinostat + immature DC (imDC) group, and 0, 50, 100 nmol/L Belinostat mature DC (mDC). The changes of the ultrastructure of DC were observed by transmission electron microscope (TEM). Immunophenotype and CCR7 expression rate were detected by FCM, and the migration rate was observed by chemotaxis assay. The proliferation of lymphocytes stimulated by different DC was detected by mixed lymphocyte culture reaction. The cytokines in the culture supernatant, including TNF-α, IL-12 and IL-10, were examined by ELISA. RQ-PCR was used to examine the relative expression of mRNA in RelB. Results: Successful cultured and identified the qualified imDC and mDC. Belinostat decreased the expression of CCR7 on imDC [(25.82±7.25)% vs (50.44±5.61)% and (18.71±2.00)% vs (50.44±5.61)%], meanwhile increased the rate on mDC [(71.14±1.96)% vs (64.90±1.47)%]. Chemotaxis assay showed that the migration rate of Belinostat+imDC and Belinostat+mDC group were both decreased, but the difference in imDC was not significant. T lymphocyte proliferation rate stimulated by 100 nmol/L Belinostat+imDC group was lower than imDC group in condition irritation cell∶reaction cell=1∶2 [(227.09±13.49)% vs (309.49±53.69)%]. Belinostat significantly suppressed the secretion of cytokines TNF-α, IL-12 and IL-10 (all P<0.01). The relative expression of mRNA in RelB was slightly decreased in Belinostat+imDC and Belinostat+mDC group (all P<0.05). Conclusion: Belinostat could effectly suppress DC maturation and regulate immune tolerance of DC, which may be due to the down-regulation of mRNA level of RelB in DC.
Animals ; Cells, Cultured ; Dendritic Cells ; Histone Deacetylase Inhibitors ; Hydroxamic Acids ; Mice ; Mice, Inbred C57BL ; Sulfonamides

We examined 216 Escherichia coli (E. coli) isolated from chickens and environmental specimens from hatcheries between 2005 and 2006 in order to evaluate the epidemiological prevalence of avian pathogenic E. coli (APEC) in Korea tentatively by multiplex PCR. The multiplex PCR which was used as tentative criteria of APEC targets 8 virulence-associated genes; enteroaggregative toxin (astA), increased serum survival protein (iss), iron-repressible protein (irp2), P fimbriae (papC), aerobactin (iucD), temperature-sensitive hemagglutinin (tsh), vacuolating autotransporter toxin (vat), and colicin V plasmid operon (cva/cvi) genes. The number of detected genes could be used as a reliable index of their virulence. It was demonstrated that E. coli strains already typed as APEC always harbor 5 to 8 genes, but non-APEC strains harbor less than 4 genes. Assuming the criteria of APEC is a possession of more than 5 virulenceassociated genes, we discriminated 24 APEC strains among the 216 E. coli strains. Contamination rates of APEC in the field were 31.3% in layers, 14.0% in broilers, 2.7% in broiler breeders, and 0.0% in environmental specimens from hatcheries. The combinational tendency of APEC examined is a fundamental possession of astA, iss and iucD genes and addition of cva/cvi, tsh, vat, and irp2 genes which have a critical importance for virulent traits of APEC. Compared with intravenous chicken challenge or embryo lethality assay, multiplex PCR method could be useful to discriminate APEC rapidly for convenient diagnosis.
Chickens ; Colicins ; Embryonic Structures ; Escherichia ; Escherichia coli ; Hemagglutinins ; Hydroxamic Acids ; Korea ; Multiplex Polymerase Chain Reaction ; Operon ; Plasmids ; Prevalence

PURPOSE: Childhood headache is different from adulthood headache and according to their age in clinical aspects. This study investigated the clinical differences of primary headache according to ages of children and adolescents. METHODS: A 300 children who did not show abnormalities on neurologic examination or brain CT or MRI were classified into two groups according to their ages. RESULTS: The percentage of those in the migraine group (24.2% vs. 35.9% in Groups 1 and 2 respectively) was higher in Group 2, but it was not statistically significant. In relation to the duration of headache, pain lasting for less than one hour accounted for 59.8% and 40% in Groups 1 and 2, respectively (P=0.001). In relation to the location headaches developed, the frontal region (40.2%) and temporal region (48.1%) were the most common in Groups 1 and 2, respectively (P<0.001). In relation to the nature of the headaches, tightening sensation accounted for the highest percentage in both groups; however, pulsating sensation were more common in Group 2 than in Group 1 (16.2% vs. 8.3%, P=0.038). In relation to the severity of headaches, severe to profound headaches accounted for 35.5% and 61.1% in Groups 1 and 2, respectively (P<0.001). In relation to laterality, unilateral headaches accounted for 12.4% and 26.7% in Groups 1 and 2, respectively (P=0.002). In relation to accompanying symptoms, the incidence of photophobia was higher in Group 2 than in Group 1 (P=0.047). CONCLUSION: Age factors should be considered in the diagnosis of childhood headaches. Also, we consider that there may be a need to establish diagnostic criteria specifically for childhood headaches separately from those for adulthood headaches.
Adolescent ; Age Factors ; Brain ; Child ; Headache ; Headache Disorders, Primary ; Humans ; Hydroxamic Acids ; Incidence ; Migraine Disorders ; Neurologic Examination ; Photophobia ; Sensation

OBJECTIVETo investigate the effect of trichostatin A(TSA) on SGC- 7901 cells.

METHODSCytotoxicity and cell viability of gastric cancer cell line SGC- 7901 were assayed by MTT method. Morphologic assessment of apoptosis was performed with fluorescence microscope. Cell cycle and apoptosis rate were analyzed by flow cytometry. Histone H3 acetylation was detected by Western blot.

RESULTSTSA showed apparently cytotoxicity in SGC- 7901 cells. The growth curve showed the growth ratio decreased with the increase of TSA concentration. Apoptosis rate were significantly different between TSA treated group(75 ng/ml for 72 h)and control group (P < 0.05). Morphologic changes of apoptosis including nuclear chromatin condensation and fluorescence strength were observed with fluorescence microscope.TSA treatment (75 ng/ml for 72 h) sensitively induced apoptosis in the cell,which was demonstrated by the migration of many cells to the sub- G1 phase,the reduction of G1- phase cells and the increment of apoptosis rate (29.54%) in flow cytometric analysis. The expression of acetylated histone H3 was increased in TSA group(75 ng/ml) for 48 h compared with control group by Western blot.

CONCLUSIONSTSA can induce SGC- 7901 cell apoptosis. The expression of acetylated histone H3 may contribute to the apoptosis.

Acetylation ; drug effects ; Apoptosis ; drug effects ; Cell Line, Tumor ; Histones ; metabolism ; Humans ; Hydroxamic Acids ; pharmacology ; Stomach Neoplasms

In order to examine the strong anticancer action and low toxicity of Trichostatin A (TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/ PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dose-dependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.
Acetylation ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; HL-60 Cells ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; chemistry ; Humans ; Hydroxamic Acids ; pharmacology

OBJECTIVETo investigate the trichostain A (TSA)-induced expression of costinmulatory molecules CD80 and CD86 in HL-60, K562 and mononuclear cells (MNC) of bone marrow in AML patients and its clinical significance.

METHODSThe TSA-induced expression of costimulatory molecules CD80, CD86 in HL-60, K562 and BMMNC, and the cell viability were detected by flow cytometry; the mRNA expression of CD80 and CD86 was detected by RT-PCR; after the TSA-induced HL-60 cells and K562 cells were irradiated with 75 Gy, the effect of these cells on proliferation of PBMNC from healthy volunteers was determined with CCK-8 method.

RESULTSThe HL-60 cells and BMMNC in AML patients expressed CD86, not expressed CD80, while the K562 cells not expressed CD86 and CD80. TSA could up-regulate the expression of CD86 in HL-60 cells and BMMNC of AML patients. The TSA-induced HL-60 cells expressing costimulatory molecule CD86 showed the proliferative effect on BMMNC from healthy volunteers.

CONCLUSIONThe TSA can induce the expression of costimulatory molecule CD86 in HL-60 cells and BMMNC in AML patients, and can improve the proliferation of PBMNC in healthy volunteers.

B7-1 Antigen ; B7-2 Antigen ; Cell Line, Tumor ; Cell Survival ; Flow Cytometry ; Humans ; Hydroxamic Acids ; Leukemia, Myeloid, Acute

OBJECTIVETo establish a rapid, relatively quantitative method of detecting acetylated proteins.

METHODSThe proteins of Jurkat cells were acetylated by Trichostatin A (TSA) at different concentrations, then enriched and purified by anti-acetylated lysine antibodies affinity chromatography colum. The components eluted by acid were fixed on the microplate, the levels of acetylated proteins were tested by ELISA, and their components were identified by MALDI-TOF-TOF mass spectrometry. Also the above-mentioned methods were applied to the other three agents (gallic acid, emodin and monoacetylated emodin A).

RESULTSThat 4 × 10(5) Jurkat cells treated with 1 µmol/L TSA produced the optimal acetylated effect, up to 22 acetylated proteins were identified by MALDI-TOF-TOF, of them 15 were acetylated histones. The other three agents also induced acetylation, the relative values of acetylated proteins of Jurkat cells treated with 35.09 µmol/L and 17.54 µmol/L gallic acid were 4.3% and 14.2% respectively; those as of 28.7% and 11.5% treated with 1.47 µmol/L and 2.94 µmol/L emodin; those as of 22.0% and 3.6% treated with 152.91 µmol/L and 30.58 µmol/L monoacetylated emodin A.

CONCLUSIONThe method based on affinity chromatography colum may be useful for the detection of acetylated proteins, and could be used to screen agents which target to histone deacetylase.

Acetylation ; Chromatography, Affinity ; Histones ; analysis ; Humans ; Hydroxamic Acids ; pharmacology ; Jurkat Cells ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

In order to examine the strong anticancer action and low toxicity of Trichostatin A (TSA), the effect of TSA was examined on the growth inhibition, acetylation of histone H3 and apoptosis in HL-60 cells by employing MTT, immunocytochemical techniques, and Annexin-V-FITC/ PI assay. Our results showed that TSA could inhibit proliferation of HL- 60 cells in a time- and dose-dependent manner, and the IC50 at the 36th h was 100 ng/ml. The apoptosis-inducing effect of TSA on HL-60 cells was also time- and dose-dependent. But it didn't demonstrate apparent apoptosis induction in NPBMNCs within specific dose and time range. Both of the acetylation of histone H3 in HL-60 cells and NPBMNCs increased significantly (P<0.05) after treated with 100 ng/ml TSA for 4 h. However, there was no significant differences between the two groups (P>0.05). It is concluded that TSA can inhibit growth and induce apoptosis of HL-60 cells in a time- and dose-dependent manner, and is able to selectively induce apoptosis in HL-60 cells but does not respond in NPBMNCs under the same conditions. The difference of TSA between HL-60 cells and NPBMNCs can't be explained by the regulation of histone acetylation.
Acetylation ; Antineoplastic Agents/pharmacology ; Apoptosis/*drug effects ; HL-60 Cells ; Histone Deacetylases/antagonists & inhibitors ; Histone Deacetylases/*chemistry ; Hydroxamic Acids/*pharmacology