Imidazo[1,2-a]Pyridine Derivatives as Novel Dual-Target Inhibitors of ABCB1 and ABCG2 for Reversing Multidrug Resistance.

ABCB1 and ABCG2 are the important ATP-binding cassette (ABC) transporters associated with multidrug resistance (MDR). Herein, we designed a series of imidazo[1,2-a]pyridine derivatives as dual-target inhibitors of ABCB1 and ABCG2 through the scaffold hopping strategy. Compound Y22 displayed potential efflux function inhibitory toward both ABCB1 and ABCG2 (reversal fold: ABCB1 = 8.35 and ABCG2 = 2.71) without obvious cytotoxicity. Y22 also enhanced the potency of antiproliferative drugs in vitro. Mechanistic studies demonstrated that Y22 slightly suppressed ATPase activity but did not affect the protein expression of ABCB1 or ABCG2. Notably, Y22 exhibited negligible CYP3A4 inhibition and enhanced the antiproliferative activity of adriamycin in vivo by restoring the sensitivity of resistant cells. Thus, Y22 may be effective clinically in combination with common chemotherapy agents. In summary, Y22 is a potential dual-target inhibitor that reverses MDR by blocking the efflux function of ABCB1 and ABCG2.

Prevention of polycystic ovary syndrome and postmenopausal osteoporosis by inhibiting apoptosis with Shenling Baizhu powder compound.

Objective: Shenling Baizhu powder (SBP) has been shown to reverse the abnormal expression of the aromatic hydrocarbon receptor (AHR) mediated by air pollution. Our study aimed to understand the main ingredient of SBP and investigate its action mechanism in preventing polycystic ovary syndrome (POCS) and postmenopausal osteoporosis (PMO). Methods: The active ingredients of SBP with the highest binding affinity to AHR were screened using a Chinese medicine database, and their binding mechanism was simulated using molecular dynamics simulation (MDS). Rutin was utilized to treat ovarian granulosa cell lines and osteoblast cell lines. The cell lines were treated with a gradient of rutin concentration (0.01 mmol/L, 0.05 mmol/L and 0.1 mmol/L) to find the optimal drug dose. PCR was used to detect AHR and apoptosis-related proteins, and WB to detect the expression of AHR, caspase-3 and cleaved-caspase-3. Finally, the CCK-8 cell proliferation assay detected the proliferation of cells. Results: We obtained Rutin through the Chinese medicine database, and dynamics simulation determined its binding sites. Ovarian granulosa cell lines and osteoblast cell lines were treated with Rutin. RT-PCR and western blotting revealed that the expression of apoptosis-associated protein Bcl-2 was elevated, and the expression of AHR, Bax, caspase-3 and PARP were decreased. CCK-8 results showed accelerated proliferation in both cell types. Conclusion: Rutin, the main ingredient of SBP compound, works by binding to AHR, which can improve POCS and PMO by inhibiting cell apoptosis and by promoting cell proliferation.

Comparison between laparoscopic uncut Roux-en-Y and Billroth II with Braun anastomosis after distal gastrectomy: A meta-analysis.

BACKGROUND: Conventional Billroth II (BII) anastomosis after laparoscopic distal gastrectomy (LDG) for gastric cancer (GC) is associated with bile reflux gastritis, and Roux-en-Y anastomosis is associated with Roux-Y stasis syndrome (RSS). The uncut Roux-en-Y (URY) gastrojejunostomy reduces these complications by blocking the entry of bile and pancreatic juice into the residual stomach and preserving the impulse originating from the duodenum, while BII with Braun (BB) anastomosis reduces the postoperative biliary reflux without RSS. Therefore, the purpose of this study was to compare the efficacy and safety of laparoscopic URY with BB anastomosis in patients with GC who underwent radical distal gastrectomy. AIM: To evaluate the value of URY in patients with GC. METHODS: PubMed, Embase, Web of Science, Cochrane Library, Chinese National Knowledge Infrastructure, Wanfang, Chinese Biomedical Database, and VIP Database for Chinese Technical Periodicals (VIP) were used to search relevant studies published from January 1994 to August 18, 2021. The following databases were also used in our search: Clinicaltrials.gov, Data Archiving and Networked Services, the World Health Organization International Clinical Trials Registry Platform Search Portal (https://www.who.int/clinical-trials-registry-platform/the-ictrp-search-portal), the reference lists of articles and relevant conference proceedings in August 2021. In addition, we conducted a relevant search by Reference Citation Analysis (RCA) (https://www.referencecitationanalysis.com). We cited high-quality references using its results analysis functionality. The methodological quality of the eligible randomized clinical trials (RCTs) was evaluated using the Cochrane Risk of Bias Tool, and the non-RCTs were evaluated using the Newcastle-Ottawa scale. Statistical analyses were performed using Review Manager (Version 5.4). RESULTS: Eight studies involving 704 patients were included in this meta-analysis. The incidence of reflux gastritis [odds ratio = 0.07, 95% confidence interval (CI): 0.03-0.19, P < 0.00001] was significantly lower in the URY group than in the BB group. The pH of the postoperative gastric fluid was lower in the URY group than in the BB group at 1 d [mean difference (MD) = -2.03, 95%CI: (-2.73)-(-1.32), P < 0.00001] and 3 d [MD = -2.03, 95%CI: (-2.57)-(-2.03), P < 0.00001] after the operation. However, no significant difference in all the intraoperative outcomes was found between the two groups. CONCLUSION: This work suggests that URY is superior to BB in gastrointestinal reconstruction after LDG when considering postoperative outcomes.

GM-CSF-miRNA-Jak2/Stat3 Signaling Mediates Chemotherapy-Induced Cancer Cell Stemness in Gastric Cancer.

Chemotherapy serves as the first choice in clinic to treat advanced gastric cancer. However, emerging evidence indicated the induction of drug resistance and cancer stem cells occasionally by chemotherapy, which seriously limit the therapeutic effects, but the regulatory mechanism remains unclear. Here we treated two human gastric cancer cell lines SGC7901 and BGC823 with 5-Fluorouracil (5-Fu) or Cisplatin (DDP) in vitro. The survived cells showed significant increase of drug resistance, cell stemness and cytokine GM-CSF expression and secretion. As such, GM-CSF was applied to stimulate gastric cancer cells, followed by the subpopulation of CD133 + CSC analysis, sphere formation assay and stemness genes expression analysis. As a result, CSCs showed induction by GM-CSF treatment. A gastric cancer animal model further indicated that the gastric cancer cells significantly promoted tumor growth after GM-CSF treatment in vivo. High-throughput miRNA and mRNA sequencing analyses identified a subset of miRNAs and mRNAs under regulation of both 5-Fu and GM-CSF in gastric cancer cells, including upregulation of miR-877-3p and downregulation of SOCS2. Targeted overexpression or knockdown of miR-877-3p in gastric cancer cells revealed the oncogenic function of miR-877-3p in regulating gastric cancer by suppressing target gene SOCS2. Jak2/Stat3 signaling pathway, as a downstream target of SOCS2, showed activation in vitro and in vivo after treatment with miR-877-3p or GM-CSF. Our findings not only revealed a novel mechanism through which chemotherapy induced CSCs in gastric cancer via GM-CSF-miRNA-Jak2/Stat3 signaling, but also provided an experimental evidence for appropriate dose reduction of adjuvant chemotherapy in treatment of cancer patients.

[Internal circadian clock and liver metabolism].

Circadian clock is an internal autonomous time-keeping system, including central clocks located in the suprachiasmatic nucleus (SCN) and peripheral clocks. The molecular circadian clock consists of a set of interlocking transcriptional-translational feedback loops that take the clock-controlled genes 24 h to oscillate. The core mechanism of molecular circadian clock is that CLOCK/BMAL1 dimer activates the transcription of cryptochromes (CRYs) and Periods (PERs), which act as transcriptional repressors of further CLOCK/BMAL1-mediated transcription. In addition to this basic clock, there is an additional sub-loop of REV-ERBα and RORα regulating the transcription of BMAL1. Approximately 80% protein-coding genes demonstrate significant rhythmicity. The earth rotation is responsible for the generation of the daily circadian rhythms. To coordinate metabolic balance and energy availability, almost all organisms adapt to the rhythm. Studies have shown that circadian clock integrating with metabolic homeostasis increases the efficiency of energy usage and coordinates with different organs in order to adapt to internal physiology and external environment soon. As the central organ of metabolism, the liver performs various physiological activities nearly all controlled by the circadian clock. There are multiple interactive regulation mechanisms between the circadian clock and the regulation of liver metabolism. The misalignment of metabolism with tissue circadian is identified as a high-risk factor of metabolic diseases. This article reviews the recent studies on circadian physiological regulation of liver glucose, lipid and protein metabolism and emphasizes oscillation of mitochondrial function. We also take an outlook for new methods and application of circadian clock research in the future.

Control of axillary bud growth in tobacco through toxin gene expression system.

The control of axillary bud development after removing the terminal buds (topping) of plants is a research hotspot, and the control of gene expression, like switching on and off, allows us to further study biological traits of interest, such as plant branching and fertility. In this study, a toxin gene control system for plants based on dexamethasone (DEX) induction was constructed, and the positive transgenic tobacco exhibited growth retardation in the application area (axillary bud). The expression level of the lethal Diphtheria toxin A (DTA) gene under different DEX concentrations at different application days was analyzed. The highest expression levels appeared at 5 days after the leaf injection of DEX. The DTA transcripts were induced by 5 µM DEX and peaked in response to 50 µM DEX at 5 days after leaf injection. Here, a chemical induction system, combined with a toxin gene, were used to successfully control the growth of tobacco axillary buds after topping. The DTA expression system under DEX induction was sensitive and efficient, therefore, can be used to control axillary bud growth and development in tobacco.

Efficacy and safety of intraoperative radiotherapy in rectal cancer: A systematic review and meta-analysis.

BACKGROUND: In recent years, intraoperative radiotherapy (IORT) has been increasingly used for the treatment of rectal cancer. However, the efficacy and safety of IORT for the treatment of rectal cancer are still controversial. AIM: To evaluate the value of IORT for patients with rectal cancer. METHODS: We searched PubMed, Embase, Cochrane Library, Web of Science databases, and conference abstracts and included randomized controlled trials and observational studies on IORT vs non-IORT for rectal cancer. Dichotomous variables were evaluated by odds ratio (OR) and 95% confidence interval (CI), hazard ratio (HR) and 95%CI was used as a summary statistic of survival outcomes. Statistical analyses were performed using Stata V.15.0 and Review Manager 5.3 software. RESULTS: In this study, 3 randomized controlled studies and 12 observational studies were included with a total of 1460 patients, who are mainly residents of Europe, the United States, and Asia. Our results did not show significant differences in 5-year overall survival (HR = 0.80, 95%CI = 0.60-1.06; P = 0.126); 5-year disease-free survival (HR = 0.94, 95%CI = 0.73-1.22; P = 0.650); abscess (OR = 1.10, 95%CI = 0.67-1.80; P = 0.713), fistulae (OR = 0.79, 95%CI = 0.33-1.89; P = 0.600); wound complication (OR = 1.21, 95%CI = 0.62-2.36; P = 0.575); anastomotic leakage (OR = 1.09, 95%CI = 0.59-2.02; P = 0.775); and neurogenic bladder dysfunction (OR = 0.69, 95%CI = 0.31-1.55; P = 0.369). However, the meta-analysis of 5-year local control was significantly different (OR = 3.07, 95%CI = 1.66-5.66; P = 0.000). CONCLUSION: The advantage of IORT is mainly reflected in 5-year local control, but it is not statistically significant for 5-year overall survival, 5-year disease-free survival, and complications.

Effects of hemoglobin variants HbJ Bangkok, HbE, HbG Taipei, and HbH on analysis of glycated hemoglobin via ion-exchange high-performance liquid chromatography.

BACKGROUND: To explore the effects of HbJ Bangkok, HbE, HbG Taipei, and α-thalassemia HbH on the results of HbA1c assessment using ion-exchange high-performance liquid chromatography (IE-HPLC). METHODS: We enrolled five patients in which the results of the IE-HPLC HbA1c assay were inconsistent with the average levels of FBG. We performed hemoglobin capillary (Hb) electrophoresis using whole-blood samples. We also sequenced the genes encoding Hb using dideoxy-mediated chain termination and analyzed HbA1c using borate affinity HPLC (BA-HPLC) and turbidimetric inhibition immunoassay (TINIA). RESULTS: Two patients had the HbJ Bangkok variant. Hb genotypes of these patients were ß41-42 /ßJ Bangkok and ßN /ßJ Bangkok , and the content of HbJ Bangkok was 93.9% and 52.4%, respectively. The remaining three patients had the following: HbE (ßN /ßE Hb genotype, 23.6% HbE content), HbG Taipei (ßN /ßG Taipei Hb genotype, 39.4% HbG Taipei content), and α-thalassemia HbH (6.1% HbH content, 2.8% Hb Bart's content). In the patients with ß-thalassemia and HbJ Bangkok variants, the presence of the variants interfered with the results of HbA1c analyses using IE-HPLC and TINIA; in the remaining four patients, there was interference with the results of HbA1c IE-HPLC but not with the TINIA assay. There was no interference with BA-HPLC HbA1c results. CONCLUSIONS: HbJ Bangkok, HbE, HbG Taipei Hb, and α-thalassemia HbH disease cause varying degrees of interference with the analysis of HbA1c using IE-HPLC. In these patients, we suggest using methods free from such interference for the analysis of HbA1c and other indicators to monitor blood glucose levels.

Evidence that growth hormone can improve mitochondrial function in oocytes from aged mice.

Decline in successful conception decreases more rapidly after 38 years of age owing to follicular depletion and decreased oocyte quality. However, limited information is available regarding the underlying mechanism and the useful treatment. This study aimed to evaluate the effects of growth hormone supplementation on oocyte maturation in vivo in aged and young mice and to determine its effect on mitochondrial function. The influence of three different doses of recombinant human growth hormone (rhGH) (0.4, 0.8 and 1.6 mg/kg/day) for 8 weeks before ovarian stimulation was analyzed. Superovulated oocytes were released from the oviduct of 12-week-old and 40-week-old female C57BL/6J mice 14-16 h after administration of human chorionic gonadotropin. Ovarian follicle and morphological analysis and oocyte maturation parameters were then evaluated. This study is the first, to our knowledge, to report that medium- and high-dose rhGH significantly increases antral follicles in aged mice but anti-Müllerian hormone (AMH) levels. Furthermore, derived oocytes, MII-stage oocyte rate, ATP levels, mitochondrial membrane potential and frequencies of homogeneous mitochondrial distribution increased. In contrast, in both aged and young mice, the mtDNA copy numbers per oocyte were similar before rhGH administration, and upon saline administration, they did not differ significantly. We conclude that medium-dose rhGH supplementation before standard ovarian stimulation regimens improves oocyte quality in aged mice, probably by enhancing mitochondrial functionality.

[Protective Effect of Schisandra Extract on Embryotoxicity and Reproductive Toxicity in Early Pregnant Rats Exposed to Benzo [a] pyrene].

OBJECTIVE: To observe protective effects of Schisandra extract (SE) on embryotoxicity and reproductive toxicity of early pregnant rats exposed to Benzo[a]pyrene (Bap). METHODS: Pregnant rat model was prepared using periodic screening cage method. Totally 50 female pregnant SD rats were divided into five groups by randomized block design according to the weight, i.e., the BaP model group, the low dose SE group, the middle dose SE group, the high dose SE group, the normal control group, 10 rats in each group. Rats in the BaP model group were administered with BaP at a daily dose of 2 mg/kg by gastrogavage. Rats in low, middle, and high dose SE groups were administered by gastrogavage with BaP (at a daily dose of 2 mg/kg) plus SE at a daily dose of 40, 200, and 1 000 mg/kg, respectively. Equal volume of olive oil was administered to rats in the normal control group by gastrogavage. All medication was performed for 8 successive days. Changes of rat body weight in each period were observed. The uterus embryonic total quality and ovary quality were measured, and organ index calculated. The number of corpus luteum, the number of embryo implantation, and the number of absorbed embryo were statistically calculated respectively. The implantation rate and the absorbed embryos rate were calculated. Serum levels of human chorionic gonadotrophin ß (ß-HCG) and progesterone (PROG) were detected by ELISA. RESULTS: Compared with the normal control group, the weight of 9-day pregnant rats, the number of embryo implantation, the uterus embryonic total index, ovary index, serum levels of ß-HCG and PROG all decreased in the Bap model group with significant difference (P < 0.05, P < 0.01). Compared with the Bap model group, body weight, the uterus embryonic total index, and the PROG level increased in 3 dose SE groups (P < 0.05, P < 0.01). Ovary index and serum ß-HCG increased in middle and high dose SE groups (P < 0.05, P < 0.01). The number of implantation obviously increased in the high dose SE groups (P < 0.01). CONCLUSION: SE could reduce the embryotoxicity and reproductive toxicity of early pregnant rats exposed to Benzo[a]pyrene.

Outcome of laparoscopy-guided hysteroscopic tubal catheterization for infertility due to proximal tubal obstruction.

STUDY OBJECTIVE: To determine pregnancy outcomes after laparoscopy-guided hysteroscopic tubal catheterization and to report its role in the era of in vitro fertilization. DESIGN: Clinical cases series (Canadian Task Force classification II-3). SETTING: Reproductive surgery center. PATIENTS: Patients with unilateral or bilateral proximal tubal obstruction as the only cause of infertility were included. INTERVENTIONS: Laparoscopy-guided hysteroscopic tubal catheterization. MEASUREMENTS AND MAIN RESULTS: Only the first spontaneous conception was considered. Cumulative conception rate (CCR) was calculated using Kaplan-Meier survival analysis. Of 168 women included, 107 (63.7%) had bilateral proximal obstruction and 61 (36.3%) had unilateral obstruction. The successful recanalization rate was 54.2% per tube and 61.9% per patient. In the 93 patients in whom at least 1 fallopian tube was successfully recanalized, 40 spontaneous pregnancies (43.0%) occurred within 24 months, of which 35 (37.6%) were intrauterine pregnancies and 28 (30.1%) resulted in live births. The CCR was 37.6% at 1 year and 43.7% at 2 years. Patients with unilateral obstruction in whom cannulation was successful had the highest CCR (60.7% at 2 years). CONCLUSION: Successful tubal cannulation led to significant improvement in the pregnancy rate, which suggests that women with a proximal tubal block could be considered for laparoscopy-guided hysteroscopic cannulation, which is still a viable alternative to in vitro fertilization.

Does ambient air pollutants increase the risk of fetal loss? A case-control study.

OBJECTIVE: To examine the associations between the ambient air pollution and early fetal loss. STUDY DESIGN: A retrospective case-control study was conducted. 959 fetal losses and 959 normal intrauterine pregnancies within 14 weeks of pregnancy in 15 general or obstetrics and gynecology hospitals were selected into case and control groups, respectively. Data based on hospital records and national pollution monitor station records were collected. Logistic regression model was conducted to examine the associations between 4 ambient air pollutants (SO2, PM10, NO2 and TSP) exposures and fetal loss. RESULTS: The ratio of fetal loss to termination of pregnancy for heating months (2.28 %) was significantly (P < 0.001) higher than that for the non-heating months (1.77 %). Logistic regression suggested that fetal loss within 14 weeks was associated with higher exposure to SO2 (OR = 19.76, 95 % CI 2.34-166.71) and TSP (OR = 2.04, 95 % CI 1.01-4.13) in the first month of pregnancy. CONCLUSION: Exposure to high levels of SO2 and TSP during the first month of pregnancy was associated with an increased risk of fetal loss in early pregnancy.

[Related factors associated with pelvic adhesion and its influence on fallopian tube recanalization in infertile patients].

OBJECTIVE: To investigate factors with pelvic adhesions and the effect of different degrees pelvic adhesions on fallopian tube recanalization in infertile patients. METHODS: Total of 527 infertile patients undergoing hysteroscopy and laparoscopic surgery in Affiliated Hospital of Chinese People's Armed Police Forces Logistics College were studied retrospectively. According to the extent of pelvic adhesions, tubal umbrella adhesions and atresia, 377 cases were classified into adhesion groups, including 73 cases in grade I, 221 cases in grade II, 75 cases in grade III and 8 cases in grade IV based on adhesion score. The 150 cases with no obvious pelvic adhesion were matched as control group. Among 8 cases with grade IV ahesion were exluded from ahesion group the relationship between pelvic adhesions and related history, abdominal lesions, tubal patency and the prognosis were studied. RESULTS: (1) Related factors: the frequency of pelvic adhesion and more than 7 years of infertility of 23.9% (88/369) in adhesion group were significantly higher than 12.0% (18/150) in control groups. (2) HISTORY: compared with the control group (12.7%, 19/150; 28.7%, 43/150; 11.3%, 17/150; 12.0%, 18/150; 17.3%, 26/150), patients with pelvic adhesions present more incidence abortion (23.6%, 87/369), uterine cavity operation (38.2%, 141/369), ectopic pregnancy (20.9%, 77/369), pelvic inflammatory disease (25.5%, 94/369) and abdominopelvic surgery (31.4%, 116/369). (3) Endoscopy exploration: the incidence of hydrosalpinx (24.7%, 91/369), tube distorted (15.7%, 58/369) and salpingostomy (72.9%, 269/369) in adhesion group were higher than those in control group (2.0%, 3/150; 4.0%, 6/150; 12.0%, 18/150), but relatively lower incidence of pelvic endometriosis lesions (5.7%, 21/369) and mesosalpinx cysts (16.3%, 60/369) than those in control group (16.0%, 24/150; 30.0%, 45/150). The rate of proximal tubal recanalization (59.5%, 91/153) in adhesion group was lower than 75.4% (52/69) in control group. However, the rate of distant tubal recanalization of 84.4%, (281/333) in adhesion group and; 13/15 in control group didn't show statistical difference. (4) PROGNOSIS: the rate of ectopic pregnancy of 9.7% (29/299) in adhesion group was significantly higher than 3.1% (4/128) in control group. Among cases with grade III adhesion exhibited the highest rate of ectopic pregnancy (13.0%, 7/54; OR = 4.62, 95%CI: 1.29 - 16.50). (5) Multivariate analysis: it was found that more than two drug abortions (OR = 3.29, 95%CI: 1.34 - 8.07), pelvic and(or) abdominal surgery history (OR = 2.20, 95%CI: 1.35 - 3.57) and pelvic inflammatory disease history (OR = 1.54, 95%CI: 1.21 - 1.97) were risk factors with pelvic adhesions. CONCLUSION: More than or equal to two drug abortion history, pelvic inflammatory disease and pelvic and abdominal surgery damage were important factors for pelvic adhesions of infertility patients, which may decrease the possibility of proximal tubal recanalization and increase ectopic pregnancy risk.

[Bioinformatic prediction of conserved microRNAs and their target genes in eggplant (Solanum melongena L.)].

MicroRNAs (miRNAs), a recently discovered class of small (-21nt), non-coding, endogenous, single-stranded RNAs in eukaryotes, regulate gene expression negatively at the post-transcriptional levels depending on the extent of complementation between miRNA and mRNA. To date, a large number of miRNAs have been reported in many species, but none for eggplant (Solanum melongena L.). In this paper, a computational homology search approach based on the conservation of miRNA sequences and the stem-loop hairpin secondary structures of miRNAs was adopted. The search was started with the known plant miRNAs compared to eggplant expressed sequence tags (EST) databases to find potential miRNAs. Following a range of filtering criteria, a total of 16 potential miRNAs belonging to 12 families were identified. Three pairs of sense and antisense strand eggplant miRNAs belonging to three different miRNA families were also found. Furthermore, miR390 and miR399 sense/antisense pairs are identified for the first time in plants. Using online software psRNATarget, we further predicted the target genes of these 16 miRNAs and got 71 potential targets genes on base of 15 eggplant miRNAs. Most of these target genes were predicted to encode proteins that play key role in eggplant growth, development, metabolism, and stress responses.

Podophyllotoxin induces CREB phosphorylation and CRE-driven gene expression via PKA but not MAPKs.

CRE-driven luciferase reporter is commonly used in drug screening systems involving G protein-coupled receptors (GPCRs). In a screen campaign designed to search for melanocortin-4 receptor (MC4R) agonists, podophyllotoxin, a microtubules disruptor, was found to induce cAMP-responsive element (CRE)-driven reporter expression. MC4R was not involved because podophyllotoxin induced CREB activation and CRE-driven transcription in cells not expressing MC4R. Previous studies indicated that intracellular calcium, PKA, and MAPKs are involved in CREB phosphorylation and activation. Our studies revealed that podophyllotoxin did not affect intracellular calcium level and the phosphorylation state of p38. Podophyllotoxin induced JNK and ERK activation, but blockade of JNK and ERK activation with specific inhibitors had no effect on podophyllotoxin-induced CREB activation and CRE-regulated gene expression. Further experiments revealed that H89, a specific inhibitor of PKA, significantly inhibited podophyllotoxin-induced CREB activation. Podophyllotoxin itself did not alter intracellular cAMP level. Taken together, podophyllotoxin induces CREB activation and CRE-driven gene expression via PKA activation by a cAMP-independent mechanism.

[Construction of RNA recombinant vector expressing STAT3 short hairpin and its effects on proliferation and apoptosis of SiHa cells].

AIM: To investigate the effects of eukaryotic vector expressing short hairpin RNA (shRNA) of signal transducers and activators of transcription 3 (STAT3) on the proliferation and apoptosis of SiHa cells. METHODS: shRNA templates were designed based on STAT3 gene sequence and cloned into pSilencer2.1-U6-neo vector. The resultant plasmid was transfected into SiHa cells with Lipofectamine 2000. The STAT3 protein and mRNA were detected by Western blot and RT-PCR, respectively. The cellular growth activity was assayed by MTT and the apoptosis was tested by flow cytometry. RESULTS: The plasmid pSilencer2.1-U6-neo-STAT3 was successfully constructed and transfected into SiHa cells. The expression of STAT3 in SiHa cells decreased, the cellular growth activity decreased, and the cell apoptosis increased. CONCLUSION: The siRNA expressing plasmid pSilencer2.1-U6-neo-STAT3 can inhibit cellular proliferation and induce the apoptosis of cervical cancer cells by suppressing the expression of STAT3.

[SSS method for screening cDNA library].

A quick and simple method subsection screening (SSS) method for screening cDNA library by PCR was established. With this method, cDNA phage plate was cut into several blocks and a couple of primers was designed according to target gene. And then the target genes were obtained by screening cDNA library. Comparing with other methods,this method has many advantages such as controlled range and clear target,and also quick and simple for obtaining the target genes. It is possible to get a target gene in one week in general by this method. Thus,the CHI gene, F3'H gene, HSP gene and one HSP partial fragment, were obtained respectively in half month in our lab. We can get twice the result with half the effort when we screen several genes in the same time. It is also suitable to screen other libraries.

[Cloning and sequence analysis of MYB transcriptional regulator SmP gene of Saussurea medusa Maxim].

A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors.

[Expression and significance of heat shock protein 70, 90 in endometrial carcinomas].

AIM: To explore the expression and significance of heat shock protein (HSP)70, 90 in endometrial carcinomas. METHODS: Expresssions of HSP70 and HSP90 in 30 normal endometrium, 30 endometrial hyperplasia and 53 endometrial carcinomas were detected by Envision immunohistochemical staining and imaging pattern analyser. RESULTS: Gray Scale value(GS) of HSP70 in endometrial carcinoma being (209.06+/-5.36) GS was obviously higher than that in normal endometrium (145.21+/-4.09)GS, P<0.01 and endometrial hyperplasia(148.59+/-4.23)GS, P<0.01. GS of HSP90 in endometrial carcinoma being (166.98+/-5.71)GS was markedly lower than that in normal endometrium (208.57+/-3.14)GS,P<0.05 and endometrial hyperplasia (249.73+/-4.94)GS, P<0.01. Expression of HSP70 increased with increased pathological grading of tumor P<0.01, whereas HSP90 expression decreased with increased pathological grading P<0.01. Expression of HSP70 in nonendometrioid carcinoma (229.90+/-3.77)GS was higher than in endometrioid carcinoma (198.37+/-3.15)GS,P<0.01 and the expression pattern of HSP90(140.21+/-3.22)was contrary to that of HSP70 (176.59+/-2.79)GS,P<0.01. There was no correlations among expression of HSP70, 90 in endometrial carcinoma and depth invaded into muscular layer, clinical stage and lymph node metastasis. CONCLUSION: Both HSP70 and HSP90 may have relation to the genesis and prognosis of endometrial carcinoma.