OBJECTIVETo study the effect of fleroxacin (FLRX) on biological properties of Bloom (BLM) helicase catalytic core (BLM642-1290 helicase) in vitro and the molecular mechanism of interaction between the two molecules.

METHODSDNA-binding and unwinding activities of BLM642-1290 helicase were assayed by fluorescence polarization and gel retardation assay under conditions that the helicase was subjected to different concentrations of FLRX. Effect of FLRX on helicase ATPase activity was analyzed by phosphorus-free assay based on a colorimetric estimation of ATP hydrolysis-produced inorganic phosphate. Molecular mechanism of interaction between the two molecules was assayed by ultraviolet and fluorescence spectra.

RESULTSThe DNA unwinding and ATPase activities of BLM642-1290 helicase were inhibited whereas the DNA-binding activity was promoted in vitro. A BLM-FLRX complex was formed through one binding site, electrostatic and hydrophobic interaction force. Moreover, the intrinsic fluorescence of the helicase was quenched by FLRX as a result of non-radioactive energy transfer. The biological activity of helicase was affected by FLRX, which may be through an allosteric mechanism and stabilization of enzyme conformation in low helicase activity state, disruption of the coupling of ATP hydrolysis to unwinding, and blocking helicase translocation on DNA strands.

CONCLUSIONFLRX may affect the biological activities and conformation of BLM642-1290 helicase, and DNA helicase may be used as a promising drug target for some diseases.

DNA ; metabolism ; Fleroxacin ; pharmacology ; Nucleic Acid Synthesis Inhibitors ; pharmacology ; RecQ Helicases ; antagonists & inhibitors ; metabolism ; Spectrometry, Fluorescence ; Spectrophotometry, Ultraviolet

PURPOSE: To compare the histopathologic and morphologic findings of encapsulated blebs following Ahmed glaucoma valve implantation and primary standard trabeculectomy with mitomycin-C. METHODS: We reviewed the records of patients with otherwise uncontrollable glaucoma who had undergone Ahmed glaucoma valve implantation or trabeculectomy with mitomycin-C. Five eyes that underwent Ahmed valve implantation and three eyes that underwent trabeculectomy needed surgical revision of the initial surgery due to encapsulated bleb development with total loss of function. The surgically removed encapsulated blebs were analyzed macroscopically and microscopically. RESULTS: Removal of the encapsulated bleb was performed at a mean follow-up time of 26.6 +/- 19.4 weeks in the Ahmed valve implantation group and 12.0 +/- 11.4 weeks in the trabeculectomy group. The fibrotic wall of the encapsulated blebs had an overall thickness of 2.48 +/- 0.42 mm in the Ahmed valve implantation group and 1.62 +/- 0.37 mm in the trabeculectomy group. Macroscopically, the coconut flesh-like smooth surface was split into two layers, and the wall of the capsule was thicker in the Ahmed valve implantation group than in the trabeculectomy group. Histopathologically, the fibrotic capsule was composed of an inner fibrodegenerative layer and an outer fibrovascular layer, and there were no histopathological differences between the two groups. CONCLUSIONS: The fibrotic capsule wall was thicker in the Ahmed valve group, but there were no differences in histological findings between the two groups.
Adult ; Blister/*surgery ; Female ; Glaucoma/physiopathology/*surgery ; *Glaucoma Drainage Implants ; Humans ; Infant ; Male ; Middle Aged ; Mitomycin/*therapeutic use ; Nucleic Acid Synthesis Inhibitors/*therapeutic use ; Reoperation ; *Trabeculectomy ; Treatment Outcome

Adenine ; analogs & derivatives ; therapeutic use ; Drug Combinations ; Hepatitis B e Antigens ; blood ; Hepatitis B, Chronic ; drug therapy ; Humans ; Nucleic Acid Synthesis Inhibitors ; therapeutic use ; Nucleosides ; antagonists & inhibitors ; biosynthesis ; Organophosphonates ; therapeutic use ; Thymidine ; analogs & derivatives ; therapeutic use

Actin cytoskeleton has been known to control and/or be associated with chondrogenesis. Staurosporine and cytochalasin D modulate actin cytoskeleton and affect chondrogenesis. However, the underlying mechanisms for actin dynamics regulation by these agents are not known well. In the present study, we investigate the effect of staurosporine and cytochalasin D on the actin dynamics as well as possible regulatory mechanisms of actin cytoskeleton modulation. Staurosporine and cytochalasin D have different effects on actin stress fibers in that staurosporine dissolved actin stress fibers while cytochalasin D disrupted them in both stress forming cells and stress fiber-formed cells. Increase in the G-/F-actin ratio either by dissolution or disruption of actin stress fiber is critical for the chondrogenic differentiation. Cytochalasin D reduced the phosphorylation of cofilin, whereas staurosporine showed little effect on cofilin phosphorylation. Either staurosporine or cytochalasin D had little effect on the phosphorylation of myosin light chain. These results suggest that staurosporine and cytochalasin D employ different mechanisms for the regulation of actin dynamics and provide evidence that removal of actin stress fibers is crucial for the chondrogenic differentiation.
Actin Cytoskeleton/*drug effects ; Actins/metabolism ; Animals ; Cell Differentiation/*drug effects ; Cells, Cultured ; Chickens ; Chondrogenesis/*drug effects ; Cytochalasin D/*pharmacology ; Mesoderm/cytology/drug effects ; Myosin Light Chains/metabolism ; Nucleic Acid Synthesis Inhibitors/*pharmacology ; Phosphorylation ; Staurosporine/*pharmacology ; Stress Fibers/drug effects

OBJECTIVETo explore the effect and the molecular targets of anti-hepatitis B virus (HBV) by beta-L-D4A in vitro.

METHODS2.2.15 cells were cultured and treated with various concentrations of beta-L-D4A for 6 hours, then the effect of anti-HBV was examined by Southern blot and the replicating core particles from the cells were isolated. The endogenous polymerase reaction and activity gel experiment were performed to monitor the activities of the DNA polymerase and reverse transcriptase.

RESULTSThe replication of HBV DNA was inhibited in a dose-dependent manner. The endogenous polymerase reaction showed both the two enzymatic activities were irreversibly inactivated in a concentration -dependent manner, with IC50 at 0.51 micromol/L and 0.55 micromol/L, respectively. But the activities of DNA polymerase and reverse transcriptase were found to remain active by activity gel with exogenous templates.

CONCLUSIONSThe mechanism of inhibiting HBV replication by beta-L-D4A may be in that either the DNA replication priming is blocked or the elongation of DNA chain is terminated irreversibly.

Antiviral Agents ; pharmacology ; DNA, Viral ; biosynthesis ; Dideoxyadenosine ; analogs & derivatives ; chemistry ; pharmacology ; Dose-Response Relationship, Drug ; Hepatitis B virus ; drug effects ; enzymology ; physiology ; Humans ; Liver Neoplasms ; pathology ; Nucleic Acid Synthesis Inhibitors ; Tumor Cells, Cultured ; Virus Replication ; drug effects

PURPOSE: To determine the efficacy of mitomycin C in reducing the recurrence of anterior urethral stricture after internal optical urethrotomy (IOU). MATERIALS AND METHODS: This was a randomized controlled trial conducted in the Department of Urology at the Institute of Kidney Diseases Peshawar from March 2011 to December 2013. A total of 151 patients who completed the study were divided into two groups by the lottery method. Group A (cases) comprised 78 patients in whom mitomycin C 0.1% was injected submucosally in the stricture after conventional IOU. Group B (controls) comprised 73 patients in whom IOU only was performed. Self-clean intermittent catheterization was not offered in either group. All patients were regularly followed up for 18 months. Recurrence was diagnosed by use of retrograde urethrogram in all patients and flexible urethroscopy in selected cases. Data were collected on a structured pro forma sheet and were analyzed by SPSS. RESULTS: The mean age of the patients in group A was 37.31+/-10.1 years and that in group B was 40.1+/-11.4 years. Recurrence of urethral stricture was recorded in 11 patients (14.1%) in group A and in 27 patients (36.9%) in group B (p=0.002). The mitomycin group also showed a delay in recurrence compared with the control group (p=0.002). CONCLUSIONS: Recurrence of urethral stricture is high after optical urethrotomy. Mitomycin C was found to be highly effective in preventing the recurrence of urethral stricture after IOU.
Adult ; Follow-Up Studies ; Humans ; Injections, Intralesional ; Male ; Middle Aged ; Mitomycin/*administration & dosage ; Nucleic Acid Synthesis Inhibitors/*administration & dosage ; Recurrence ; Time Factors ; Urethral Stricture/*prevention & control/radiography/surgery ; Urodynamics ; Urologic Surgical Procedures/methods

Chemokine KC has been considered to be a murine homologue of human GRO/MGSA and was identified as chemoattractant for monocytes and neutrophils. This study examined the expression of KC mRNA in thioglycollate-elicited mouse peritoneal macrophages that were stimulated in vitro with Candida albicans (CA). Also examined were the inhibitory effects of IL-10 on the CA-induced expression of KC gene by Northern blot analysis. CA was found to induce chemokine gene expression in a gene-specific manner, CXC chemokine IP-10 mRNA expression was not detected in CA-stimulated macrophages. Maximum KC mRNA expression was observed approximately 2 hr after adding CA. The inhibitory action of IL-10 to CA-induced KC mRNA expression on mouse peritoneal macrophages was independent on concentration and stimulation time of IL-10 and was observed approximately one hour after adding IL-10 and CA simultaneously. IL-10 produced a decrease in the stability of KC mRNA, and CA-stimulated macrophages with cycloheximide blocked the suppressive effect of IL-10. These results suggest that CA also induces chemokine KC from macrophages, and IL-10 acts to destabilize CA-induced KC mRNA and de novo synthesis of an intermediate protein is a part of the IL-10 suppressive mechanism.
Animal ; Blotting, Northern ; Candida albicans/metabolism* ; Cells, Cultured ; Chemotactic Factors/genetics* ; Dactinomycin/pharmacology ; Dose-Response Relationship, Drug ; Gene Expression Regulation/drug effects* ; Growth Substances/genetics* ; Interleukin-10/pharmacology* ; Interleukin-10/metabolism ; Macrophages/physiology* ; Mice ; Mice, Inbred BALB C ; Nucleic Acid Synthesis Inhibitors/pharmacology ; RNA, Messenger/metabolism ; RNA, Messenger/drug effects

OBJECTIVETo investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.

METHODSA549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.

RESULTSThe expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.

CONCLUSIONDRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.

20-Hydroxysteroid Dehydrogenases ; genetics ; metabolism ; Alpha-Amanitin ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Dichlororibofuranosylbenzimidazole ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Nucleic Acid Synthesis Inhibitors ; pharmacology

Catechins, components of green tea, reduce the incidence of cardiovascular diseases such as atherosclerosis. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMC), resulting in atherosclerosis. The acting mechanisms of the catechins remain to be defined in the proliferation of VSMC induced by Ang II. Here we report that catechin, epicatechin (EC), epicatechingallate (ECG) or epigallocatechingallate (EGCG) significantly inhibits the Ang II-induced [3H]thymidine incorporation into the primary cultured rat aortic VSMC. Ang II increases the phosphorylation of the extracellular signal-regulated protein kinase 1/2 (ERK 1/2), c-jun-N-terminal kinase 1/2 (JNK 1/2), or p38 mitogen-activated protein kinases (MAPKs) and mRNA expression of c-jun and c-fos. The EGCG pretreatment inhibits the Ang II-induced phosphorylation of ERK 1/2, JNK 1/2, or p38 MAPK, and the expression of c-jun or c-fos mRNA. U0126, a MEK inhibitor, SP600125, a JNK inhibitor, or SB203580, a p38 inhibitor, attenuates the Ang II-induced [3H]thymidine incorporation into the VSMC. In conclusion, catechins inhibit the Ang II-stimulated VSMC proliferation via the inhibition of the Ang II-stimulated activation of MAPK and activator protein-1 signaling pathways. The antiproliferative effect of catechins may be associated with the reduced risk of cardiovascular diseases by the intake of green tea. Catechins may be useful in the development of prevention and therapeutics of vascular diseases.
Signal Transduction/drug effects ; Rats, Sprague-Dawley ; Rats ; RNA, Messenger/metabolism ; Proto-Oncogene Proteins c-jun/metabolism ; Proto-Oncogene Proteins c-fos/metabolism ; Phosphorylation ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Muscle, Smooth, Vascular/cytology/*drug effects ; Mitogen-Activated Protein Kinases/*metabolism/physiology ; Female ; DNA/biosynthesis ; Cells, Cultured ; Cell Proliferation/*drug effects ; Cell Culture Techniques ; Catechin/analogs & derivatives/*pharmacology/physiology ; Animals ; Angiotensin II/*pharmacology

Apoptotic cell death induced by p53 occurs at a late G1 cell cycle checkpoint termed the restriction(R)point, and it has been proposed that p53-induced apoptosis causes upregulation of CD95. However, as cells with defective in CD95 signaling pathway are still sensitive to p53-induced apoptosis, CD95 cannot be the sole factor resulting in apoptosis. In addition, unlike p53-induced apoptosis, the relationship between CD95-mediated apoptosis and the cell cycle is not clearly understood. It would there-fore be worth investigating whether CD95-mediated cell death is pertinent with p53-induced apoptosis in view of cell cycle related molecules. In this report, biochemical analysis showed that etoposide-induced apoptosis caused the induction and the nuclear translocation of effector molecules involved in G1 cell cycle checkpoint. However, there was no such translocation in the case of CD95-mediated death. Thus, although both types of apoptosis involved caspase activation, the cell cycle related proteins responded differently. This argues against the idea that p53-induced apoptosis occurs through the induction of CD95/CD95L expression.
Active Transport, Cell Nucleus ; Antigens, CD95/*metabolism ; *Apoptosis ; Cell Cycle ; Cell Nucleus/metabolism ; Coculture ; Dose-Response Relationship, Drug ; Down-Regulation ; Etoposide/pharmacology ; Flow Cytometry ; Human ; Immunoblotting ; Jurkat Cells ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Protein Binding ; Protein Transport ; Protein p53/*metabolism ; Signal Transduction ; Up-Regulation