Temporal Distribution Patterns of Cryptic Brachionus calyciflorus (Rotifera) Species in Relation to Biogeographical Gradient Associated with Latitude.

Sympatric distribution and temporal overlap of cryptic zooplankton species pose a challenge to the framework of the niche differentiation theory and the mechanisms allowing competitor coexistence. We applied the methods of phylogenetic analysis, DNA taxonomy, and statistical analysis to study the temporal distribution patterns of the cryptic B. calyciflorus species, an excellent model, in three lakes, and to explore the putative mechanisms for their seasonal succession and temporal overlap. The results showed that in the warm-temperate Lake Yunlong, B. fernandoi and B. calyciflorus s.s. underwent a seasonal succession, which was largely attributed to their differential adaptation to water temperature. In the subtropical Lake Jinghu, B. fernandoi, B. calyciflorus s.s., and B. dorcas exhibited both seasonal succession and temporal overlap. Seasonal successions were largely attributed to their differential adaptation to temperature, and temporal overlap resulted from their differential responses to algal food concentration. In the tropical Lake Jinniu, B. calyciflorus s.s. persisted throughout the year and overlapped with B. dorcas for 5 months. The temporal overlap resulted from their differential responses to copepod predation. These results indicated that the temporal distribution pattern of the cryptic B. calyciforus species and the mechanism that allows competitor coexistence vary with different climate zones.

Functional trait divergence associated with heteromorphic leaves in a climbing fig.

Introduction: Plants that display heteroblasty possess conspicuous variations in leaf morphology between their juvenile and adult phases, with certain species retaining juvenile-like leaves even in adulthood. Nevertheless, the ecological advantages of maintaining two or more distinct leaf types in heteroblastic plants at the adult stage remain unclear. Method: The aim of this study is to examine the adaptive significance of heteroblastic leaves sampled from branches with divergent functions (sterile and fertile branches) of mature Ficus pumila individuals by comparing their morphological, anatomical, and physiological characteristics. Result: Leaves on sterile branches (LSs) exhibited a significantly larger specific leaf area, thinner palisade and spongy tissues, lower chlorophyll contents, and lower light saturation points than leaves on fertile branches (LFs). These results demonstrate that LSs are better adapted to low light environments, while LFs are well equipped to take advantages of high light conditions. However, both LFs and LSs have a low light compensation point with no significant difference between them, indicating that they start to accumulate photosynthetic products under similar light conditions. Interestingly, significant higher net photosynthetic rate was detected in LFs, showing they have higher photosynthetic capacity. Furthermore, LFs produced significant more nutrients compared to LSs, which may associate to their ability of accumulating more photosynthetic products under full light conditions and higher photosynthetic capacity. Discussion: Overall, we observed a pattern of divergence in morphological features of leaves on two functional branches. Anatomical and physiological features indicate that LFs have an advantage in varied light conditions, providing amounts of photosynthetic products to support the sexual reproduction, while LSs adapt to low light environments. Our findings provide evidence that heteroblasty facilitates F. pumila to utilize varying light environments, likely associated with its growth form as a climbing plant. This strategy allows the plant to allocate resources more effectively and optimize its overall fitness.

Orexin receptor type 2 agonism inhibits thermogenesis in brown adipose tissue by attenuating afferent innervation.

Orexin signaling has been associated with energy expenditure and brown adipose tissue (BAT) function. However, conflicting data exist in the field about how orexin signaling regulates BAT thermogenesis. In this study, we show that a specific orexin receptor type 2 (OX2R) agonist [Ala11, D-Leu15]-OxB (OB-Ala) inhibited intrascapular brown adipose tissue (iBAT) thermogenesis by reducing sympathetic output to iBAT. This effect is mediated by OX2Rs located on afferent nerve endings innervating iBAT instead of brown adipocyte itself. Microinjection of OB-Ala into iBAT inhibited iBAT thermogenesis in mice upon cold exposure and neuronal activity in the paraventricular nucleus. Findings suggest that OB-Ala could inhibit iBAT thermogenesis by attenuating sensory input thereby inhibiting the sympathetic-sensory iBAT feedback loop. Our study uncovers a novel primary action site of orexin in the regulation of energy balance.

Efficacy of Two-dimensional Scanning Digital Kymography in Evaluation of Atrophic Vocal Folds.

OBJECTIVE: The purpose of this study was to evaluate the clinical feasibility and diagnostic accuracy of two-dimensional scanning digital kymography (2D DKG) in patients with vocal cord atrophy before and after treatment. MATERIALS AND METHODS: We analyzed the characteristics of vocal fold vibration in five patients with unilateral vocal fold paralysis and five patients with presbyphonia. In patients with vocal cord paralysis, the status before and after intracordal injection was compared. Furthermore, in patients with presbyphonia, we compared the status before and after voice therapy (Seong-Tae Kim's laryngeal calibration technique). Quantitative parameters such as amplitude and phase symmetry indices, jitter, shimmer, noise-to-harmonic ratio, and maximum phonation time and qualitative parameters such as Voice Handicap Index, glottal gap, amplitude, and phase difference were used to evaluate the pre- and post-treatment status. RESULTS: In cases of vocal cord paralysis, vibratory changes of the vocal folds before and after intracordal injection could be identified immediately using 2D DKG. In overcorrection cases, all of the measured parameters were poor except for improvement of the glottal gap. In addition, 2D DKG showed appropriately the changes in vocal cord vibration before and after voice therapy in patients with presbyphonia. CONCLUSION: Two-dimensional DKG may be a useful diagnostic tool in evaluation of the vibratory characteristics of entire vocal cords. In addition, it may also play a role in providing a decision for treatment modalities.

The efficacy and safety of long- versus short-interval transarterial chemoembolization in unresectable hepatocellular carcinoma.

Background: To compare the efficacy and safety of long- versus short-interval of transarterial chemoembolization (TACE) in unresectable hepatocellular carcinoma (HCC) patients. Methods: This retrospective analysis enrolled 574 patients with unresectable HCC who underwent at least two sessions of TACE between January 2007 and December 2014. The patients were divided into a short-interval group (SIG) and a long-interval group (LIG) based on the median TACE interval of the first two sessions. Propensity score matching (PSM) identified 476 patients for a comparison of overall survival (OS) and safety. Results: Before matching, the LIG had a longer OS than the SIG (Median: 12.1 vs. 8.7 months; P = 0.003). After matching, median OS in the SIG and LIG were 9.1 and 14.2 months (P < 0.001). The 1-, 2-, and 3-year survival rates were 37.5%, 17.1%, and 9.9% for SIG and 50.1%, 19.3%, and 11.6% for LIG, respectively. The TACE interval was an independent prognostic factor for OS. The LIG had a longer OS than the SIG in Barcelona Clinic liver cancer (BCLC) stage C patients (Median: 10.2 vs. 5.8 months; P < 0.001), but not in BCLC-A or B. The postoperative adverse rates were similar in matched SIG and LIG patients (29.4% vs. 33.6%, P = 0.324). Conclusions: A long interval between the first two sessions of TACE resulted in a better OS than a short interval in patients with unresectable BCLC C-stage HCC.

Emergence agitation during recovery from intracranial surgery under general anaesthesia: a protocol and statistical analysis plan for a prospective multicentre cohort study.

INTRODUCTION: Emergence agitation after intracranial surgery is an important clinical issue during anaesthesia recovery. The aim of this multicentre cohort study is to investigate the incidence of emergence agitation, identify the risk factors and determine clinical outcomes in adult patients after intracranial surgery under general anaesthesia. Additionally, we will deliberately clarify the relationship between postoperative pneumocephalus and agitation. METHODS AND ANALYSIS: The present study is a prospective multicentre cohort study. Five intensive care units (ICUs) in China will participate in the study. Consecutive adult patients admitted to the ICUs after intracranial surgery will be enrolled. Sedation-Agitation Scale (SAS) or Richmond Agitation-Sedation Scale (RASS) will be used to evaluate the patients 12 h after the enrolment. Agitation is defined as an SAS score of 5-7, or an RASS score of +2 to +4. According to the maximal SAS and RASS score, patients will be divided into two cohorts: the agitation group and the non-agitation group. Factors potentially related to emergence agitation will be collected at study entry, during anaesthesia and operation, during postoperative care. Univariate analyses between the agitation and the non-agitation groups will be performed. The stepwise backward logistic regression will be carried out to identify the independent predictors of agitation. Patients will be followed up for 72 h after the operation. Accidental self-extubation of the endotracheal tube and removal of other catheters will be documented. The use of sedatives and analgesics will be collected. ETHICS AND DISSEMINATION: Ethics approval has been obtained from each of five participating hospitals. Study findings will be disseminated through peer-reviewed publications and conference presentations. TRIAL REGISTRATION NUMBER: NCT02318199.

Gene silencing of ß-catenin by RNAi inhibits proliferation of human esophageal cancer cells by inducing G0/G1 cell cycle arrest.

OBJECTIVES: The aim of the present study was to explore mechanisms underlying the effects of down-regulating ß-catenin expression on esophageal carcinoma (EC) cells. METHODS: Cell cycle distribution and apoptosis were determined using flow cytometry and annexin V apoptosis assay, respectively. Transmission electron microscopy (TEM) was used to examine changes in ultrastructure, while expression of cyclin D1 protein and mRNA was detected by western blot and real-time PCR. Proliferating cell nuclear antigen (PCNA) and extracellular signal-regulated kinase (ERK) 1/2 were evaluated by Western blot analysis. PCNA labeling index (LI) was determined by immunocytochemistry. RESULTS: Compared with pGen-3-con transfected and Eca-109 cells, the percentage of G0/G1-phase pGen-3-CTNNB1 transfected cells was obviously increased (P<0.05), with no significant difference among the three groups with regard to apoptosis (P>0.05). pGen-3-CTNNB1 transfected cells exhibited obvious decrease in cyclin D1 mRNA and protein expression (P<0.05) and the ultrastructure of Eca-109 cells underwent a significant change after being transfected with pGen-3-CTNNB1, suggesting that down-regulating ß-catenin expression can promote the differentiation and maturation. The expression of PCNA and the ERKI/2 phosphorylation state were also down-regulated in pGen-3-CTNNB1 transfected cells (P<0.05). At the same time, the PCNA labeling index was decreased accordingly (P<0.05). CONCLUSION: Inhibition of EC Eca-109 cellproliferation by down-regulating ß-catenin expression could improve cell ultrastructure by mediating blockade in G0/G1 through inhibiting cyclin D1, PCNA and the MAPK pathway (p-ERK1/2).

Involvement of profilin-1 in angiotensin II-induced vascular smooth muscle cell proliferation.

Profilin-1, a regulator of actin polymerization, has recently been linked to vascular hypertrophy and remodeling. Whether profilin-1 is involved in angiotensin (Ang) II-induced proliferation of vascular smooth muscle cells leading to vascular remodeling in hypertension remains unclear. The present study was designed to analyze the correlation of profilin-1 and vascular remodeling during hypertension and to evaluate the role of profilin-1 in proliferation of vascular smooth muscle cells and the underlying mechanisms. The vascular morphology and the expression of profilin-1 in arterial tissues of spontaneously hypertensive rats and Wistar-Kyoto rats were assessed. The profilin-1 expression was significantly increased concomitantly with definite vascular remodeling by evaluating the media thickness, lumen diameter, media thickness-to-lumen diameter ratio and mean nuclear area in artery media in spontaneously hypertensive rats, which was inhibited by treatment with losartan. In cultured rat aortic smooth muscle cells (RASMCs), Ang II induced profilin-1 expression in a dose- and time-dependent manner. Knockdown of profilin-1 using small hairpin RNA inhibited Ang II-induced proliferation of RASMCs. Moreover, blockade of JAK2/STAT3 signaling pathway also inhibited Ang II-induced proliferation of RASMCs and profilin-1 expression. These results suggest that profilin-1 mediates the proliferation of RASMCs induced by Ang II via activation of Ang II type 1 receptor/JAK2/STAT3 signaling pathway, which may contribute to vascular remodeling in hypertension.

Exposure to trimethoprim/sulfamethoxazole but not other FDA category C and D anti-infectives is associated with increased risks of preterm birth and low birth weight.

OBJECTIVE: To examine the association between trimethoprim/sulfamethoxazole, other US Food and Drug Administration (FDA) C and D anti-infectives, and non anti-infective FDA C, D, and X drugs used during pregnancy with preterm birth and low birth weight. METHODS: We carried out a retrospective cohort study based on a 50% random sample of women who gave birth in the Canadian province of Saskatchewan from 1997 to 2000. The association between trimethoprim/sulfamethoxazole, other FDA C and D anti-infectives (fluconazole, clarithromycin, doxycycline, and tetracycline), and non anti-infective FDA C, D, and X drugs used during pregnancy with preterm birth and low birth weight was evaluated using multiple logistic regression, with adjusted odds ratios (aORs) and 95% confidence intervals (CIs) as association measures. RESULTS: A total of 17 939 women were included in the final analysis. Trimethoprim/sulfamethoxazole was associated with significantly increased risks for preterm birth (aOR 1.51, 95% CI 1.10, 2.08) and low birth weight (aOR 1.67, 95% CI 1.14, 2.46). Exposure to non anti-infective FDA category C, D and X drugs was also associated with increased risks for preterm birth (aOR 1.17, 95% CI 1.09, 1.31) and low birth weight (aOR 1.14, 95% CI 0.92, 1.42), but to a lesser degree. Other FDA C and D anti-infectives were not (statistically) significantly associated with increased risks for preterm birth (aOR 0.93, 95% CI 0.49, 1.77) or low birth weight (aOR 0.65, 95% CI 0.27, 1.60). CONCLUSIONS: Among FDA C, D and X drugs, trimethoprim/sulfamethoxazole, a folic acid antagonist, has the strongest association with preterm birth and low birth weight.

Effect of asymmetric dimethylarginine on MIF expression and TNF-α and IL-8 secretion in THP-1 monocytes-derived macrophages.

OBJECTIVE: To investigate the effect of ADMA on macrophage migration inhibitory factor (MIF) expression and tumor necrosis factor-α (TNF-α) and IL-8 secretion in THP-1 monocyte-derived macrophages. METHIDS: THP-1 monocytes were induced to differentiate into macrophages by a 24-h incubation with 160 nmol/L PMA. The THP-1 monocyte-derived macrophages were exposed to different concentrations of ADMA for 24 h, and the changes in MIF mRNA and protein expressions were analyzed with RT-PCR and Western blotting, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of TNF-α and IL-8 in the supernatant of THP-1-derived macrophages following ADMA treatments. RESULTS: ADMA obviously up-regulated MIF mRNA and protein expressions in THP-1-derived macrophages in a concentration- dependent manner. Exposure of the cells to 15 µmol/L ADMA for 24 h showed the most potent effect in up-regulating MIF mRNA and protein expressions. ADMA treatment also resulted in a dose-dependent increase of the levels of TNF-α and IL-8 in the culture supernatant of the macrophages, and the peak levels occurred following the treatment with 15 µmol/L ADMA. CONCLUSION: ADMA can up-regulate MIF expression and induce TNF-α and IL-8 secretion in THP-1 monocyte-derived macrophages.

Left atrial wall tension directly affects the restoration of sinus rhythm after Maze procedure.

OBJECTIVE: The surgical treatment of permanent atrial fibrillation (AF) continues to be a clinical challenge. Increased left atrial (LA) wall tension affected by LA wall thickness and volume has been associated with poor restoration of sinus rhythm after the Maze procedure. This study was designed to investigate the impact of conversion rates to sinus rhythm using LA wall tension reduction techniques in conjunction with aggressive postoperative pharmacological therapy in comparison to a modified Maze procedure alone. METHODS: From 1999 to 2008, 322 patients with permanent AF and biatrial enlargement, who required mitral valve ± tricuspid valve surgery were exactly randomized into two groups: The study group used biatrial reduction with reef-imbricate suture technique concomitant with the Maze procedure and aggressive postoperative pharmacological therapy; the control group was treated with the Maze procedure alone. LA dimension was measured by transesophageal echocardiogram (TEE) or transthoracic echocardiography (TTE); LA wall thickness was measured by TEE and manually during surgery. Pulmonary artery (PA) pressures were measured by PA catheter or TTE, BNP test and clinical follow-up at discharge, 3 months, 6 months and 1 year. There were 187 woman (58%) and 135 men (42%). Their mean age was 45 ± 9.5 years. RESULTS: Overall restoration of sinus rhythm was significantly improved in the group with aggressive reduction of LA wall tension during 1-year clinical follow-up (89.3% vs 67.2%, p < 0.001). Calculated LA wall tension was significantly reduced at discharge in the study group versus control group (4.012 ± 1.650 dyn cm(-1) vs 20 384 ± 3313 dyn cm(-1) (p < 0.001)) and at 1-year follow-up (1059 ± 1161 dyn cm(-1) vs 17 139 ± 3170 dyn cm(-1) (p < 0.001)), respectively. Significant differences in changes in LA dimension were detected at discharge and 1-year follow-up in the study group versus control group (43 ± 7 vs 61 ± 11, p < 0.001). LA wall (3.9 ± 1.3 vs 2.3 ± 0.9) thickness also significantly differed at the 1-year follow-up. CONCLUSION: An aggressive approach to reduce LA wall tension significantly improves restoration of sinus rhythm after the Maze procedure. LA wall tension directly affects sinus conversion. Further studies using pharmacologic intervention to reduce LA wall tension for maintenance of sinus rhythm need to be evaluated.

Asymmetric dimethylarginine upregulates the expression of ACAT-1 in THP-1 macrophage-derived foam cells.

OBJECTIVE: To investigate the effects of asymmetric dimethylarginine (ADMA) on ACAT-1 expression and cholesterol content in THP-1-derived macrophages and foam cells. METHODS: THP-1 cells were induced to differentiate into macrophages and further into foam cells. The macrophages and foam cells were exposed to different concentrations (0, 3.75, 7.5, 15, and 30 µmol/L) of ADMA for varying time lengths (6, 12, and 24 h), and the changes in ACAT-1 mRNA and protein levels in the cells were measured with RT-PCR and Western blotting. The cellular cholesterol content was measured with enzyme-linked colorimetry assay. RESULTS: In THP-1-derived macrophages and foam cells, the expression levels of ACAT-1 mRNA and protein and cellular cholesterol content increased significantly in response to ADMA treatment in a time- and concentration-dependent manner. CONCLUSION: ADMA may play an important role in inducing foam cell formation from macrophages. ACAT-1 inhibition targeting the macrophages and foam cells may serve as a potential therapeutic target in the treatment of atherosclerosis.

Recombinant proteins secreted from tissue-engineered bioartificial muscle improve cardiac dysfunction and suppress cardiomyocyte apoptosis in rats with heart failure.

BACKGROUND: Tissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 (IGF-1) protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction (MI) and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles (BAMs) following coronary artery ligation in Sprague-Dawley rats. METHODS: Ligation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 (rhIGF-1), and green fluorescent protein (GFP), and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups: MI-IGF group (n = 6) and MI-GFP group (n = 6). The MI-IGF group received rhIGF-secreting BAM (IGF-BAMs) transplantation, and the MI-GFP group received GFP-secreting BAM (GFP-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: S-IGF group (n = 6) and S-GFP group (n = 6). The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting. RESULTS: Primary rat myoblasts were retrovirally transduced to secrete rhIGF-1 and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted consistent levels of hIGF (0.4 - 1.2 µg×BAM(-1)×d(-1)). When implanted into syngeneic rat, IGF-BAMs secreted and delivered rhIGF. Four weeks after therapy, the hemodynamics was improved significantly in MI rats treated with IGF-BAMs compared with those treated with GFP-BAMs. The levels of serum IGF-1 were increased significantly in both MI and sham rats treated with IGF-BAM. The mRNA expression of bax was lower and Bcl-2 expression was higher in MI-IGF group than MI-GFP group (P < 0.05). Western blotting assay showed TNF-α and caspase 3 expression was lower in MI-IGF group than MI-GFP group after therapy. CONCLUSIONS: rhIGF-1 significantly improves left ventricular function and suppresses cardiomyocyte apoptosis in rats with chronic heart failure. Genetically modified tissue-engineered BAMs provide a method delivering recombinant protein for the treatment of heart failure.

Recombinant human growth hormone secreted from tissue-engineered bioartificial muscle improves left ventricular function in rat with acute myocardial infarction.

BACKGROUND: Experimental studies and preliminary clinical studies have suggested that growth hormone (GH) treatment may improve cardiovascular parameters in chronic heart failure (CHF). Recombinant human GH (rhGH) has been delivered by a recombinant protein, by plasmid DNA, and by genetically engineered cells with different pharmacokinetic and physiological properties. The present study aimed to examine a new method for delivery of rhGH using genetically modified bioartificial muscles (BAMs), and investigate whether the rhGH delivered by this technique improves left ventricular (LV) function in rats with CHF. METHODS: Primary skeletal myoblasts were isolated from several Sprague-Dawley (SD) rats, cultured, purified, and retrovirally transduced to synthesize and secrete human rhGH, and tissue-engineered into implantable BAMs. Ligation of the left coronary artery or sham operation was performed. The rats that underwent ligation were randomly assigned to 2 groups: CHF control group (n = 6) and CHF treatment group (n = 6). The CHF control group received non-rhGH-secreting BAM (GFP-BAMs) transplantation, and the CHF treatment group received rhGH-secreting BAM (GH-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: sham control group (n = 6) and sham treatment group (n = 6). The sham control group underwent GFP-BAM transplantation, and the sham treatment group underwent GH-BAM transplantation. GH-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats with ligation of the left coronary artery or sham operation was performed. Eight weeks after the treatment, echocardiography was performed. hGH, insulin-like growth factor-1 (IGF-1) and TNF-alpha levels in rat serum were measured by radioimmunoassay and ELISA, and then the rats were killed and ventricular samples were subjected to immunohistochemistry. RESULTS: Primary rat myoblasts were retrovirally transduced to secrete rhGH and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted 1 to 2 microg of bioactive rhGH per day. When implanted into syngeneic rat, GH-BAMs secreted and delivered rhGH. Eight weeks after therapy, LV ejection fraction (EF) and fractional shortening (FS) were significantly higher in CHF rats treated with GH-BAMs than in those treated with GFP-BAMs ((65.0 +/- 6.5)% vs (48.1 +/- 6.8)%, P < 0.05), ((41.3 +/- 7.4)% vs (26.5 +/- 7.1)%, P < 0.05). LV end-diastolic dimension (LVEDD) was significantly lower in CHF rats treated with GH-BAM than in CHF rats treated with GFP-BAM (P < 0.05). The levels of serum GH and IGF-1 were increased significantly in both CHF and sham rats treated with GH-BAM. The level of serum TNF-alpha decreased more significantly in the CHF treatment group than in the CHF control group. CONCLUSIONS: rhGH significantly improves LV function and prevents cardiac remodeling in rats with CHF. Genetically modified tissue-engineered bioartificial muscle provides a method delivering recombinant protein for the treatment of heart failure.

Gene silencing of beta-catenin by RNAi inhibits cell proliferation in human esophageal cancer cells in vitro and in nude mice.

beta-Catenin, which is frequently overexpressed in a variety of human cancers including esophageal cancer, mediates cancer cell proliferation and tumor growth. In the present study, we used a human U6 promoter-driven DNA-template approach to induce short hairpin RNA (shRNA)-triggered RNA interference to silence beta-catenin gene expression in human esophageal squamous cell carcinoma cell line Eca-109, and then evaluated its effects on the proliferation and growth of tumor cells in vitro and in nude mice. beta-Catenin expression levels decreased markedly in Eca-109 cells transfected with a plasmid expressing shRNA for beta-catenin. Downregulation of beta-catenin was concomitantly accompanied by reduction of cyclin D1, colony formation, and growth inhibition of Eca-109 cells in vitro. The mechanism appears to be the G0/G1 phase arrest but not induction of apoptosis. In vivo, treatment of Eca-109 cells with beta-catenin shRNA greatly impeded tumor growth in nude mice. We conclude that plasmid vector-mediated beta-catenin RNA interference holds great promise as a novel treatment on human esophageal cancer with beta-catenin overexpression.

Lithium inhibits proliferation of human esophageal cancer cell line Eca-109 by inducing a G2/M cell cycle arrest.

AIM: To investigate the effect of lithium on proliferation of esophageal cancer (EC) cells and its preliminary mechanisms. METHODS: Eca-109 cells were treated with lithium chloride, a highly selective inhibitor of glycogen synthase kinase 3beta (GSK-3beta), at different concentrations (2-30 mmol/L) and time points (0, 2, 4, 6 and 24 h). Cell proliferative ability was evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, and cell cycle distribution was examined by flow cytometry. Expressions of p-GSK-3beta, beta-catenin, cyclin B1, cdc2 and cyclin D1 protein were detected by Western blotting, and the subcellular localization of beta-catenin was determined by immunofluorescence. The mRNA level of cyclin B1 was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Lithium could inhibit the proliferation of Eca-109 cells. Lithium at a concentration of 20 mmol/L lithium for 24 h produced obvious changes in the distribution of cell cycle, and increased the number of cells in G(2)/M phase (P<0.05 vs control group). Western blotting showed that lithium inhibited GSK-3beta by Ser-9 phosphorylation and stabilized free beta-catenin in the cytoplasm. Immunofluorescence further confirmed that free beta-catenin actively translocated to the nucleus. Moreover, lithium slightly elevated cyclin D1 protein expression, whereas lowered the cyclin B1 expression after 24 h lithium exposure and no obvious change was observed for cdc2 protein. CONCLUSION: Lithium can inhibit the proliferation of human esophageal cancer cell line Eca-109 by inducing a G(2)/M cell cycle arrest, which is mainly mediated through the inhibition of lithium-sensitive molecule, GSK-3beta, and reduction of cyclin B1 expression.

Effects of transplanted myoblasts transfected with human growth hormone gene on improvement of ventricular function of rats.

BACKGROUND: Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human growth hormone (hGH) has been shown to promote angiogenesis and attenuate apoptosis in the experimental animal. This study was conducted to explore the effects of myoblast-based hGH gene therapy on heart function restoration and angiogenesis after myocardial infarction, and to compare the differences between myoblast-based hGH gene therapy and myoblast therapy. METHODS: Myoblasts were isolated from several SD rats, cultured, purified, and transfected with plasmid pLghGHSN and pLgGFPSN. Radioimmunoassay (RIA) was used to detect the expression of hGH in these myoblasts. SD rats underwent the ligation of the left anterior descending coronary artery so as to establish a heart ischemia model. Thirty surviving rats that underwent ligation were randomly divided into 3 equal groups 2 weeks after left coronary artery occlusion: pLghGHSN group received myoblast infected with hGH gene transplantation; pLgGFPSN group received myoblast infected with GFP gene transplantation; control group: received cultured medium only. Four weeks after the injection the surviving rat underwent evaluation of cardiac function by echocardiography. The rats were killed and ventricular samples were undergone immunohistochemistry with hematoxylin-eosin and factor VIII. Cryosection was analyzed by fluorescence microscopy to examine the expression of green fluorescent protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of vascular endothelial growth factor (VEGF), bax and Bcl-2. hGH expression in myocardium was examined by Western blot. RESULTS: Myoblast can be successfully isolated, cultured and transfected. The expression of hGH in transfected myoblast was demonstrated with RIA. Four weeks after therapy, the cardiac function was improved significantly in pLghGHSN group and pLgGFPSN group. Fractional shortening (FS) and ejection fraction (EF) in pLghGHSN group were elevated significantly compared with pLgGFPSN group and control group after therapy (FS: 36.9+/-5.3 vs 29.5+/-3.5, 21.8+/-2.9; EF: 56.9+/-4.3 vs 47.1+/-3.6, 38.4+/-4.8, P<0.05). Left ventricular end-diastolic dimension (LVEDD) and heart infracted size in pLghGHSN group were decreased significantly compared with pLgGFPSN group and control group after therapy (LVEDD: 5.9+/-0.3 vs 6.8+/-0.2, 8.6+/-0.3; heart infracted size: (34.5+/-4.2)% vs (40.0+/-3.9)%, (46.1+/-3.8)%, P<0.05); Green fluorescence was detected in cryosection of pLgGFPSN group. The capillary density of the pLgGFPSN group was significantly greater than those of the pLghGHSN group and control group (P<0.05). The mRNA expression of VEGF and Bcl-2/bax in pLghGHSN group was higher than in pLgGFPSN group or control group (P<0.05). The expression of hGH gene in myocardium tissue can be detected by Western blot assay in pLghGHSN group. CONCLUSIONS: Transplantation of heart cells transfected with hGH induced greater angiogenesis and effect of antiapoptosis than transplantation of cells transfected with GFP. Combined GH gene transfer and cell transplantation provided an effective strategy for improving postinfarction ventricular function.

Role of asymmetric dimethylarginine in inflammatory reactions by angiotensin II.

Previous investigations have demonstrated that angiotensin (Ang) II induces inflammatory reactions and asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, might be a novel inflammatory factor. Endothelial cell activation was induced by incubation with Ang II or ADMA. Incubation with Ang II (10(-6) M) for 24 h elevated the levels of ADMA and decreased the levels of nitrite/nitrate concomitantly with a significant increase in the expression of protein arginine methyltransferase and a decrease in the activity of dimethylarginine dimethylaminohydrolase (DDAH). Exposure to Ang II (10(-6) M for 24 h) also enhanced intracellular ROS elaboration and the levels of tumor necrosis factor (TNF)-alpha and interleukin (IL)-8, upregulated chemokine receptor CXCR2 mRNA expression, increased adhesion of endothelial cells to monocytes and induced a significant increase in the activity of nuclear factor (NF)-kappaB, which was attenuated by pretreatment with the Ang II receptor blocker losartan (1, 3 and 10 muM). Exogenous ADMA (30 microM) also increased ROS generation and the levels of TNF-alpha and IL-8, decreased the levels of nitrite/nitrate, upregulated CXCR2 gene expression, increased endothelial cell binding with monocytes and activated the NF-kappaB pathway, which was inhibited by pretreatment with losartan or L-arginine. These data suggest that ADMA is a potential proinflammatory factor and may be involved in the inflammatory reaction induced by Ang II.

Effects of valsartan and indapamide on plasma cytokines in essential hypertension.

OBJECTIVE: investigate and compare the effect of valsartan and indapamide on inflammatory cytokines in hypertension. METHODS: Forty-one untreated patients with mild to moderate hypertension and 20 age and sex-matched normotensives were enrolled in this study. Hypertensives were treated with indapamide 1.5 mg/d (n=20) or valsartan 80 mg/d (n=21) for 4 weeks, and blood samples for determining monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1 (MIP-1alpha), sP-selectin, asymmetric dimethylarginin (ADMA), angiotensin II (Ang II), and 6-keto-PGF1alpha were collected before the treatment and 4 weeks after the treatment. RESULTS: Hypertensives exhibited significantly higher blood pressure, as well as elevated plasma levels of MCP-1, MIP-1alpha, sP-selectin and serum level of ADMA compared with the normotensives. Nevertheless, there was no significant difference in serum 6-keto-PGF1alpha and Ang II between the hypertensives and the normotensives. After the treatment with indapamide or valsartan for 4 weeks, both the systolic and diastolic blood pressures, though still higher than those of the normotensives, decreased markedly. After the treatment with indapamide for 4 weeks, MCP-1, MIP-1alpha and sP-selectin slightly decreased, but not statistically significant (P>0.05). Those cytokines decreased significantly after being treated with valsartan for 4 weeks [(19.16+/-3.11) pg/mL vs (16.08+/-2.67) pg/mL, P<0.05; (27.74+/-8.36) pg/mL vs (17.64+/-7.59) pg/mL, P<0.05; (2.67+/-3.18) pg/mL vs (6.15+/-2.94) pg/mL, P<0.01]. In the 2 treatment groups, 6-keto-PGF1alpha markedly increased [(61.96+/-20.81) pg/mL vs (96.72+/-25.89) pg/mL, P<0.05; (63.25+/-16.92) pg/mL vs (143.22+/-43.45) pg/mL, P<0.01]; ADMA decreased significantly [(1.35+/-0.74) pg/mL vs (0.98+/-0.56) micromol/L, P<0.05; (1.31+/-0.68) pg/mL vs (0.71+/-0.52) micromol/L, P<0.01]. Though Ang II slightly increased, no statistical significance was found (P>0.05). CONCLUSION: The levels of MCP-1, MIP-1alpha, sP-selectin and ADMA were elevated in mild to moderate hypertensives. Valsartan and indapamide have similar blood pressure lowering effect. Valasartan exerts more significant effect on cytokines than indapamide does.