Antiproliferative activity of Farnesol in HeLa cervical cancer cells is mediated via apoptosis induction, loss of mitochondrial membrane potential (ΛΨm) and PI3K/Akt signalling pathway.

PURPOSE: Cervical cancer remains the most gruesome health problem in women worldwide as it ranks third in incidence. Despite recent developments in the treatment options of cervical cancer, the survival of patients not fit for surgical treatment rather remains poor. The main purpose of the current research was to determine the anticancer effect of farnesol in HeLa human cervical cancer cells together with studying its impact on apoptosis induction, mitochondrial membrane potential (MMP) and PI3K/Akt signalling cascade. METHODS: Cell viability was estimated by MTT assay while clonogenic assay was used to assess the effects on colony formation tendency in these cells. Fluorescence microscopy indicated apoptosis induction while flow cytometry showed the farnesol effects on the loss of MMP. RESULTS: Farnesol exerted both dose and time-dependent antiproliferative effects on cervical cancer cells with IC50 values of 33.5, 23.8 and 17.6 µM at 24, 48 and 72 hrs time intervals, respectively. Colony formation of HeLa cells was considerably affected in a dose-dependent manner with the addition of farnesol to the cell culture. Farnesol-treated cells mostly emitted orange fluorescence indicating apoptotic cell death and this effect increased with increasing dose of the compound. Furthermore, farnesol induced considerable reduction in the number of cells with depolarized mitochondria corresponding to a reduction of MMP. With increase in the dosage of farnesol, there was a noticeable decrease in the expression levels of PI3K, p-PI3K and p-Akt proteins. CONCLUSIONS: In brief, this study showed that farnesol -a naturally occurring sesquiterpene- exerts powerful antiproliferative activity via apoptosis induction, loss of MMP and downregulation of the expression levels of PI3K, p-PI3K and p-Akt proteins.

[Effects of environmental and biotic factors on soil respiration in a coastal wetland in the Yellow River Delta, China.] / çŽ¯å¢ƒå› å­å’Œç”Ÿç‰©å› å­å¯¹é»„æ²³ä¸‰è§’æ´²æ»¨æµ·æ¹¿åœ°åœŸå£¤å‘¼å¸çš„å½±å“.

Using the Li-8150 multichannel automatic soil CO2 efflux system, soil respiration was measured continuously over a one-year period in a coastal wetland in the Yellow River Delta, China. Environmental and biological factors were measured simultaneously, including temperature, soil water content, aboveground biomass and leaf area index. The results showed that the diurnal variation of soil respiration presented a single-peak curve, but it appeared as multiple peaks when disturbed by soil freezing and surface flooding. Soil respiration showed obvious seasonal dynamics and a single peak curve. The average annual soil respiration was 0.85 µmol CO2·m-2·s-1, and the mean soil respiration rate was 1.22 µmol CO2·m-2·s-1 during the growing season. On one-year scale, soil temperature was a major factor influencing soil respiration in the coastal wetland, which explained 87.5% of the variation in soil respiration. On the growing season scale, soil water content and leaf area index accounted for 85% of the seasonal variation of soil respiration.

Proteomic alteration of equine monocyte-derived macrophages infected with equine infectious anemia virus.

Similar to the well-studied viruses human immunodeficiency virus (HIV)-1 and simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV) is another member of the Lentivirus genus in the family Retroviridae. Previous studies revealed that interactions between EIAV and the host resulted in viral evolution in pathogenicity and immunogenicity, as well as adaptation to the host. Proteomic analysis has been performed to examine changes in protein expression and/or modification in host cells infected with viruses and has revealed useful information for virus-host interactions. In this study, altered protein expression in equine monocyte-derived macrophages (eMDMs, the principle target cell of EIAV in vivo) infected with the EIAV pathogenic strain EIAV(DLV34) (DLV34) was examined using 2D-LC-MS/MS coupled with the iTRAQ labeling technique. The expression levels of 210 cellular proteins were identified to be significantly upregulated or downregulated by infection with DLV34. Alterations in protein expression were confirmed by examining the mRNA levels of eight selected proteins using quantitative real-time reverse-transcription PCR, and by verifying the levels of ten selected proteins using parallel reaction monitoring (PRM). Further analysis of GO and Kyoto Encyclopedia of Genes and Genomes (KEGG)-Pathway enrichment demonstrated that these differentially expressed proteins are primarily related to the biological processes of oxidative phosphorylation, protein folding, RNA splicing, and ubiquitylation. Our results can facilitate a better understanding of the host response to EIAV infection and the cellular processes required for EIAV replication and pathogenesis.

Infection of equine monocyte-derived macrophages with an attenuated equine infectious anemia virus (EIAV) strain induces a strong resistance to the infection by a virulent EIAV strain.

The Chinese attenuated equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. Given that the induction of immune protection results from the interactions between viruses and hosts, a better understanding of the characteristics of vaccine strain infection and host responses would be useful for elucidating the mechanism of the induction of immune protection by the Chinese attenuated EIAV strain. In this study, we demonstrate in equine monocyte-derived macrophages (eMDM) that EIAVFDDV13, a Chinese attenuated EIAV strain, induced a strong resistance to subsequent infection by a pathogenic strain, EIAVUK3. Further experiments indicate that the expression of the soluble EIAV receptor sELR1, Toll-like receptor 3 (TLR3) and interferon ß (IFNß) was up-regulated in eMDM infected with EIAVFDDV13 compared with eMDM infected with EIAVUK3. Stimulating eMDM with poly I:C resulted in similar resistance to EIAV infection as induced by EIAVFDDV13 and was correlated with enhanced TLR3, sELR1 and IFNß expression. The knock down of TLR3 mRNA significantly impaired poly I:C-stimulated resistance to EIAV, greatly reducing the expression of sELR1 and IFNß and lowered the level of infection resistance induced by EIAVFDDV13. These results indicate that the induction of restraining infection by EIAVFDDV13 in macrophages is partially mediated through the up-regulated expression of the soluble viral receptor and IFNß, and that the TLR3 pathway activation plays an important role in the development of an EIAV-resistant intracellular environment.

An attenuated EIAV strain and its molecular clone strain differentially induce the expression of Toll-like receptors and type-I interferons in equine monocyte-derived macrophages.

Activations of endosomal TLRs include TLR3, TLR7/8, and TLR9 stimulates the production of cytokines, such as type I interferons (IFNs), and therefore involves in virus-host interactions. In the present study, two equine anemia virus (EIAV) strains EIAVFDDV13 and EIAVFDDV3-8, which showed different induction on protective immunity, were compared regarding their ability to regulate the expression of endosomal TLRs, as well as type I IFNs, after infection of equine monocyte-derived macrophages (eMDMs). Our results showed that EIAVFDDV13 dramatically up-regulated the expression of TLR3 and IFNß and less robustly up-regulated the expression of TRL9 and IFNα1, whereas EIAVFDDV3-8 induced significantly lower expression of type I IFN mRNA and protein and more strongly down-regulated the expression of TLR7 and TLR8. In addition, no significant differences in cell apoptosis were observed between these two strains. Given that the genomic variation of EIAVFDDV13 is considerably higher than that of molecular clone EIAVFDDV3-8, our results suggest that stronger TLR3 activation and increased INFß production induced by the multi-species strain are associated with an effective vaccine-elicited protective immune response.

[The application of single-genome amplification and sequencing in genomic analysis of an attenuated EIAV vaccine].

Our previous studies found that the Chinese attenuated EIAV vaccine was composed of a pool of quasispecies, which showed a complicated diversity called "multi-species". Further determining the viral composition of these species in the vaccine should improve the identification of predominant viruses in the vaccine and facilitate the analysis of in vivo evolution of EIAV and the vaccine. In this study, the comparison of fidelities in amplifying and sequencing the V3 to V5 fragment of EIAV envelope gp90 gene by either a single-genome amplification (SGA) approach or the traditional RT-PCR (bulk PCR) was performed. Results revealed that the diversities were 1.84% and 1.88% for SGA- and bulk PCR-derived sequences, respectively. Futher analysis revealed that beside the sequences highly homologous to those derived by the bulk PCR, nine of 73 sequences derived by SGA contained a deduced amino acid domain that was identical to the corresponding domain in the virulent strain LN40. In addition, sequences with deletion of one predicted amino acid residual was detected by using SGA The presence of these less populated sequences provided additional evidence for the "multi-species" hypothesis for the action mechanism of the EIAV vaccine. Furthermore, based on the analysis of sampling bias, Our results that the difference in copy number of each viral specie in the pool of quasispecies resulted in the inefficiency to amplify viral sequences that were in low population by bulk PCR. Therefore, the sequences amplified by bulk PCR could not correctly represent the composition of quasispecies. As an approach based on the amplification and sequencing single isolated genome, SGA significantly improved the weakness of bulk PCR and appeared its advantage in analysis of EIAV genome composition with high variety.

[Studying the damages of mouse retina induced by 2,5-hexanedione].

OBJECTIVE: To study toxic effects of 2,5-hexanedione (2,5-HD) on pathology and lipid peroxidation in mouse retina. METHODS: Forty-eight mice were randomly divided into blank control group (12 mice), negative control group exposed to normal solution (12 mice) and group exposed to 2,5-HD for 2. 4 and 8 weeks, respectively (24 mice) by intraperitoneal injection (2.5% 2,5-HD) at the dose of 400 mg/kg. The pathological changes of mouse retina were examined under light microscope. The activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in mouse retina were detected. RESULTS: The retinal structure in the blank and negative control groups was normal. In mice exposed to 2,5-HD for 8 weeks, the swelling of outer and inner segments and disorder arrangement of the segments without clear boundary were found. The staining of outer plexiform layers was uneven and the irregular loose structure appeared. The hyperchromatic pyknotic and necrosis nuclei were presented in ganglion cells layer. Compared with the control and blank groups, the activities of SOD gradually and significantly reduced and the concentrations of MDA increased in group exposed to 2,5-HD (P < 0.05). CONCLUSION: 2,5-HD can induce the injury of retina tissues of mice, which may be associated with the lipid peroxidation.

A multi-center study of a modified open trocar first-puncture approach in 17 350 patients for laparoscopic entry.

BACKGROUND: Laparoscopic entry is of primary importance in laparoscopic surgery because of its potential association with serious complications such as visceral and vascular injuries. There are several approaches now available for laparoscopic entry. The present study reported a modified open trocar first-puncture approach (Yan's open technique) and validated its safety and practicability in a multi-center research. METHODS: The study was performed in seven gynecological endoscopy centers for 8 successive years from September 1998 to March 2006 involving 17 350 patients, who received the modified open trocar first-puncture approach developed by Dr. LIU Yan as the study group (MOT group). The "Yan's open technique" is the umbilical incision with a scalpel and then a 10-mm trocar entry into the abdominal cavity through direct trocar puncture or insertion of the cannula sheath via the opened umbilicus under no resistance. Another 4570 patients received the traditional Veress needle puncture as the control (VN group). The first puncture procedures of both groups were performed by 28 experienced gynecologic laparoscopists and 170 learners. RESULTS: In MOT group, the successful achievement rate (AR) of first puncture was 99.99% (17 348/17 350), including smooth manipulation in 17 326 cases and unsmooth manipulation in 22 cases. The remaining two cases failed. First-puncture associated complications occurred in two cases (0.01%). In VN group, the successful AR of first puncture was 99.89% (4565/4570), including smooth manipulation in 4542 cases and unsmooth manipulation in 23 cases. The remaining five cases failed. First-puncture associated complications occurred in four cases (0.09%). There was no significant difference in the successful AR between the experienced gynecologic laparoscopists of the two groups (100% vs 100%, P > 0.05), but the difference was significant between the learners of the two groups (99.98% vs 99.81%, P < 0.05). The complication rate of VN group was significantly higher than that of MOT group (0.09% vs 0.01%, P < 0.05). CONCLUSIONS: Compared with the traditional Veress needle puncture, the modified open trocar first-puncture is easier to follow, especially for learners. In addition, it can avoid possible Veress needle-associated injuries. Opening the umbilical hole for the sake of minimizing or zeroing puncture resistance is a safer and more practicable maneuver for laparoscopic entry.

[Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA].

OBJECTIVES: To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12. METHODS: The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT. RESULTS: All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01). CONCLUSION: siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.

[Caspase-12 expression and activation in the pathogenesis of acute hepatic failure induced by lipopolysaccharide and D-galactosamine].

OBJECTIVE: To study the role of caspase-12 expression on hepatocyte apoptosis in an experimental model of acute hepatic failure (AHF). METHODS: A mouse experimental model of AHF was developed by intraperitoneal injection of lipopolysaccharide (LPS) and D-galactosamine (D-Gal). Hepatocyte apoptosis was examined by DNA agarose gel and liver pathology. Caspase-12 mRNA expression in liver was detected by reverse transcriptase PCR (RT-PCR) method. The expression of caspase-12, GRP78 proteins in livers was determined by Western blot. RESULTS: Caspase-12 mRNA expression in the livers increased significantly from 5 to 7 hours after administration of LPS and D-Gal. Typical manifestation of hepatocyte apoptosis appeared at 5 hours after the drug administration. After 5 hours the level of serum ALT and AST were remarkably increased, and they reached the peak at 7 hours. The expression of procaspase-12 protein decreased obviously at 7 hours. Seven hours after the drug administration, hepatocyte apoptosis and necrosis both started. The marker of endoplasmic reticulum (ER) stress, Bip/GRP78 was activated during the development of hepatocyte apoptosis. The level of Bip/GRP78 protein was gradually increased at 5 hours after the drug induction. CONCLUSION: Hepatocyte apoptosis plays an important role in the pathogenesis of AHF. Caspase-12 induced ER stress involves in hepatocyte apoptosis. It suggests that inhibition of caspase-12 activation might be a potential strategy in the treatment of AHF in the future.