[Expression and significance of PD-1 and PD-L1 in the specimens of epithelial ovarian cancer].

Objective: To examine the expression of programmed cell death 1 (PD-1) and its ligand (PD-L1) in epithelial ovarian cancer (EOC) tissues, and investigate the correlation among their expression, clinicopathological features and prognosis. Methods: The specimens of 180 patients with EOC treated in the First Affiliated Hospital of Dalian Medical University from October 2002 to December 2013 were confirmed by pathological examination. The pathological tissue specimens of subtypes ，included 120 cases of serous carcinoma, 30 cases of mucinous carcinoma, 20 cases of endometrioid carcinoma, and 20 cases of clear cell carcinoma. The normal paracancerous tissues of 50 cases randomly selected from the 180 patients as control group. Immunohistochemical SP method was used to detect the expressions of both PD-1 and PD-L1 in epithelial ovarian cancer tissues, and the relationships among their expressions,the clinicopathological parameters and prognosis were respectively analyzed. Results: （1） PD-1 was expressed in lymphocytes infiltrated in EOC tissues, and PD-L1 was expressed in the cell membranes of cancer tissues. In all EOC cases, 33 cases (18.3%, 33/180) of both PD-1 and PD-L1 were highly expressed, and only 1 (2.0%, 1/50) of control group showed high expression. There was statistically significant difference between two groups (P<0.01). (2) Among the four subtypes tissue specimens of EOC, the high expression rate of PD-1 was 25.0% (30/120) for serous carcinoma, 3/15 for endometrioid carcinoma, 0 (0/30) for mucinous carcinoma, and 0 (0/15) for clear cell carcinoma. The high expression rate of PD-L1 was 23.3% (28/120) for serous carcinoma, 3.3% (1/30) for mucinous carcinoma, 2/15 for endometrioid carcinoma, and 2/15 for clear cell carcinoma. Both PD-1 and PD-L1 expressions in the four sub-types of tissue specimens were significantly different (P<0.05). The high expression rate of both PD-1 and PD-L1 was 9.2% (8/87) in the early stage and 26.9% (25/93) in the late stage. There was a statistically significant difference between the two groups (P<0.01). Similarly, the expression of both PD-1 and PD-L1 were significantly higher in the cases of high-grade EOC （type Ⅱ） than those of low-grade （type Ⅰ） and in the cases of EOC distributed bilaterally than that distributed unilaterally, and there were statistically significant differences (P<0.05). (3) The Kaplan-Meier survival analysis showed that the survival time were respectively 35 and 36 months in the cases with high expressions of both PD-1 and PD-L1, and the survival time were the same as 61 months in the cases with low expression of both PD-1 and PD-L1, and the comparison was statistically significant (P<0.05). Conclusions: The expression levels of PD-1 and PD-L1 in EOC tissues are higher than those in adjacent tissues, especially in serous carcinomas. The expression of both PD-1 and PD-L1 is higher in specimens of the patients with advanced stages. The results showed that the high expression of both PD-1 and PD-L1 is an indicator of poor prognosis of patients suffering from EOC.

[Expression and significance of MAPK/ERK in the specimens and cells of epithelial ovarian cancer].

Objective: To detect phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) protein expression in epithelial ovarian cancer and cell lines, and to examine the effects of mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor AZD6244 on cell proliferation, apoptosis as well as cell cycle of ovarian cancer cells. To explore the function and significance of MAPK/extracellular signal-regulated kinase (ERK) signaling pathway in the development of ovarian cancer. Methods: (1) A total of 104 cases of patients with ovarian cancer who accepted the treatment of gynecological surgery and being confirmed by pathological examination in First Affiliated Hospital, Dalian Medical University from January 2004 to December 2013 were selected. The expressions of p-ERK1/2 protein were detected by immunohistochemistry in ovarian cancer specimens, and the relationship between the expressions of p-ERK1/2 and the clinical features of patients was analyzed. (2) p-ERK1/2 and other related proteins were determined by western blot in various ovarian cancer cells, including SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3 treated with or without MEK inhibitor. The cellular proliferation, apoptosis and cell cycle of ovarian cancer cells after treatment with MEK inhibitor were analyzed by methyl thiazolyl tetrazolium (MTT) assay and flow cytometry, respectively. Results: (1) The immunohistochemical method showed that p-ERK1/2 between low grade serous carcinoma and clear cell carcinoma were not significantly higher expressed (P>0.05) . However, a lower level of the p-ERK1/2 expression were observed among high grade serous carcinoma, mucinous carcinoma and endometrioid carcinoma (all P<0.05) . There was no significant correlation between the protein expression of p-ERK1/2 and patients' age, pathological stage of surgery, and preoperative serum CA(125) level (P>0.05). (2) Western blot showed that the protein p-ERK1/2 was widely expressed in various ovarian cancer cell lines such as SKOV3, OV2008, C13, A2780S, A2780CP, OVCAR4, OVCAR5, OVCAR8 and CAOV3. After treatment with AZD6244 (5, 10 µmol/L), the level of p-ERK1/2 in OVCAR5 and OVCAR8 decreased significantly in dose-dependent manner. Additionally, we found a reduction of the expression level of cyclin D1, caspase-3 and appeared cleaved poly adenosine diphosphate ribose polymerase (PARP) in OVCAR5 and OVCAR8, compared with control groups. MTT assays showed that OVCAR5, OVCAR8 and A2780S were differently inhibited in the dose-dependent manner after being treated with different concentrations of AZD6244 (0, 2.5, 5, 10, 25, 50 and 100 µmol/L, all P<0.05). Further tested by flow cytometry, the results showed that AZD6244 (5, 10 µmol/L) was able to induce the apoptosis of OVCAR5, OVCAR8 and A2780S, as well as G(0)/G(1) phase arrest, both in a dose-dependent manner (P<0.05). Conclusions: As the main active and functional unit of MAPK/ERK signaling pathway, p-ERK1/2 protein is expressed in both the tissues and various ovarian cancer cell lines. AZD6244 could down-regulated the expression of p-ERK1/2 in ovarian cancer cells, accompanied by the decreased proliferation and increased cell apoptosis of ovarian cancer cells. In conclusion, MAPK/ERK signaling pathway might play a role in the development and progression of ovarian cancer, and may be provide a novel option for molecular targeted therapies of the disease.

Cation complexation behavior of pyrene- and anthracene-appended new crown ether derivatives.

Pyrene- and anthracene-appended new crown ether derivatives have been synthesized by Schiff's base reaction, and cation complexation behavior was investigated by fluorescence spectroscopy measurements. Based on photo-induced electron transfer and intramolecular charge transfer mechanism, the host molecules emit stronger fluorescence in the presence of various cations Na(+), K(+), Rb(+), Cs(+) and NH(4)(+) since the complexation between guest cations and crown ether compounds inhibit partial electron transfer from the nitrogen atom to the chromophores and subsequently fluorescence is enhanced. The binding constants were estimated from the plots of the fraction of binding sites filled for crown ether compound as a function of free-ion concentration. Results show that 15-crown-5 derivatives exhibit higher binding ability with sodium cations while 18-crown-6 derivatives had higher affinity for potassium cations, which is consistent with the hole-size relationship of the crown ethers. Ammonium ion complexation caused largest fluorescence enhancement. It is understood that ammonium ion cannot only complex with crown ether, but also interact directly with the lone pair electrons of nitrogen atom in C=N bond so that electron transfer from the nitrogen atom to chromophores is further inhibited.

Detection of Toll-like receptor 2 (TLR2) mutation in the lepromatous leprosy patients.

Toll-like receptor 2 (TLR2) is critical in the immune response to mycobacterial infections and the mutations in the TLR2 have been shown to confer the susceptibility to severe infection with mycobacteria. To define this, we screened the intracellular domain of TLR2 in 131 subjects. Groups of 45 lepromatous and 41 tuberculoid leprosy (TT) patients and 45 controls were investigated. Ten subjects among the lepromatous leprosy (LL) patients had a band variant detected by single-stranded conformational polymorphism. DNA sequencing detected a C to T substitution at nucleotide 2029 from the start codon of the TLR2. The mutation would substitute Arg to Trp at amino acid residue 677, one of the conserved regions of TLR2. In our results, the mutation was involved in only LL, not TT and control. Thus, we suggest that the mutation in the intracellular domain of TLR2 has a role in susceptibility to LL.

An efficient cell-free protein synthesis system using periplasmic phosphatase-removed S30 extract.

An efficient cell-free translation system was developed by removal of phosphatase localized in the periplasmic space, which hampers the translation reaction by hydrolyzing ATP. S30 extract was prepared from the spheroplast of Escherichia coli, and as much as 40% of ATP-hydrolysis activity of phosphatases could be easily removed by the spheroplast formation. The reaction period of translation using phosphatase-removed S30 extract could be prolonged and protein synthesis was enhanced by more than 30%.

Typing of clinical isolates of Mycobacterium leprae and their distribution in Korea.

Although there is no genetic diversity in isolates of Mycobacterium leprae, the variance of tandem repeats in the rpoT gene was recently demonstrated. We have typed clinical isolates of M. leprae in Korea using difference of the tandem repeats in the rpoT gene. Among 69 patients, 65 Korean isolates (94.2%) demonstrated four copies of the 6 bp tandem repeat (GACATC) in the rpoT gene, and incidences of three copies were found in only two Koreans and two foreigners (2.9%, respectively).

A simple regression method for determining the quantity of translated protein in a cell-free protein synthesis system.

A simple regression method for quantifying endogenous leucine in S30 extract was developed. This method enabled the quantity of translated protein in a cell-free protein synthesis system to be exactly determined.

Variable (CA/GT)n simple sequence repeat DNA in the alga Chlamydomonas.

A partial genomic DNA library of Chlamydomonas reinhardtii was screened with an (AC)11 probe for the presence of (CA/GT)n simple sequence repeats (SSRs). Based on the frequency of these repeats in the partial genomic library, we estimate that (CA/GT)n repeats occur at a rate of about one every 17.7 kb in the C. reinhardtii genome. Ten positive clones were sequenced and four polymerase chain reaction (PCR) primer sets flanking (CA/GT)n sequences were constructed for four loci. The PCR was used to specifically amplify these regions from multiple isolates of C. reinhardtii. All four loci were highly polymorphic in the C. reinhardtii isolates. A simple Mendelian inheritance pattern was found for all four loci, which showed 2:2 segregation in the tetrads resulting from a cross between C. reinhardtii and C. smithii. Our results suggest that these simple sequence repeat DNA loci will be useful for identity testing, population studies, linkage analysis, and genome mapping in Chlamydomonas.

Fluorescence lifetime imaging with near-field scanning optical microscopy.

Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.

Comparison of two different PCR amplification products (the 18-kDa protein gene vs. RLEP repetitive sequence) in the diagnosis of Mycobacterium leprae.

To determine the best molecular method for diagnosing leprosy, two sets of Mycobacterium leprae-specific primers were compared. Fresh biopsies and slit skin smear samples were obtained from 67 leprosy patients and examined by touchdown (TD) PCR using primers amplifying either a 129-bp fragment of the RLEP repetitive sequence or a 360-bp fragment of the 18-kDa protein gene of M. leprae. Seventeen of 30 (56.7%) biopsy specimens and four of 37 (10.8%) slit skin smear specimens were positive using the primer for the 18-kDa protein gene, whereas 24 of 30 (80%) biopsy and 27 of 37 (73%) slit skin smear samples showed detectable PCR products in the RLEP repetitive sequence. Twenty-one of 31 cases (67.7%) with a bacterial index of zero were PCR positive for the primer RLEP repetitive sequence. These results demonstrate that detection of M. leprae using PCR with primers to a RLEP sequence is more sensitive and specific than PCR with the 18-kDa protein gene primers and also slit smears with acid fast staining. PCR of RLEP repetitive sequences is therefore a useful means of detecting M. leprae DNA even when it is present at very low levels.

Effects of molecular oxygen on multiphoton-excited photochemical analysis of hydroxyindoles.

We have examined the effects of dissolved molecular oxygen on multiphoton-excited (MPE) photochemical derivatization of serotonin (5HT) and related cellular metabolites in various buffer systems and find that oxygen has a profound effect on the formation efficiency of visible-emitting photoproducts. Previously, end-column MPE photoderivatization provided low mass detection limits for capillary electrophoretic analysis of hydroxyindoles, but relied on the use of Good's buffers to generate high-sensitivity visible signal. In the present studies, visible emission from 5HT photoderivatized in different buffers varied by 20-fold under ambient oxygen levels but less than 2-fold in the absence of oxygen; oxygen did not significantly alter the photoproduct excited-state lifetime (approximately 0.8 ns). These results support a model in which oxygen interferes with formation of visible-emitting photoproducts by quenching a reaction intermediate, an effect that can be suppressed by buffer molecules. Deoxygenation of capillary electrophoresis separation buffers improves mass detection limits for 5-hydroxyindoles fractionated in 600-nm channels by approximately 2-fold to < or =30000 molecules and provides new flexibility in identifying separation conditions for resolving 5HT from molecules with similar electrophoretic mobilities, such as the catecholamine neurotransmitters.

Influence of arachidonic acid on catecholamine secretion in the perfused rat adrenal medulla.

The present study was conducted to investigate the influence of arachidonic acid, which is known to be an important unsaturated fatty acid component of membrane phospholipids and to be liberated by phospholipase A2 action, on secretion of catecholamines (CA) from the isolated perfused rat adrenal glands and to clarify the mechanism of its action. Arachidonic acid (10 uM) perfused into an adrenal gland of the rat for 20 min caused a significant inhibition of CA secretion evoked by ACh (5.32 x 10(-3) M), DMPP (10(-4) M) and muscarine (10(-4) M) while it did not affect that induced by excess K+ (5.6 x 10(-2) M). Arachidonic acid, in the presence of ouabain (100 uM), an inhibitor of Na+, K(+) -ATPase, also produced a marked inhibitory effect of CA secretion evoked by ACh, DMPP and muscarine but did not modify the secretory effect of excess K+. The perfusion of arachidonic acid along with indomethacin (30 uM), which is an inhibitor of cyclooxygenase, for 20 min attenuated markedly CA secretory effect evoked by ACh, DMPP and muscarine while it did not influence that by excess K+. Prostaglandin F2 alpha perfused in a retrograde direction for 20 min inhibited greatly the CA secretion evoked by DMPP but did not affect the effect evoked by excess K+. All of arachidonic acid, ouabain, indomethacin and prostaglandin F2 alpha used in the present study did not affect the spontaneous basal release of CA in the perfused rat adrenal glands. Taken together, these experimental results suggest that arachidonic acid, as well as prostaglandin F2 alpha, cause the inhibitory action of CA secretion evoked by cholinergic receptor-mediated stimulation, but not by membrane depolarization, and also play a modulatory role in regulating CA secretion from the rat adrenal medulla.

Influence of pentobarbital-Na on stimulation-evoked catecholamine secretion in the perfused rat adrenal gland.

OBJECTIVES: The present study was attempted to investigate the effects of pentobarbital-Na, one of the barbiturates which are known to depress excitatory synaptic transmission in the central nervous system at concentrations similar to those required for the induction and maintenance of anesthesia, on catecholamines (CA) secretion evoked by cholinergic stimulation and membrane-depolarization from the isolated perfused rat adrenal gland, and to clarify the mechanism of its action. METHODS: Mature male Sprague-Dawley rats were anesthetized with thiopenal-Na (40 mg/kg, s.c.). The adrenal gland was isolated by the methods of Wakade. A cannula used for perfusion of the adrenal gland was inserted into the distal end of the renal vein. The adrenal gland was carefully removed from the animal and placed on a platform of a leucite chamber. RESULTS: The perfusion of pentobarbital-Na(30-300 uM) into an adrenal vein for 20 min produced relatively dose-dependent inhibition in CA secretion evoked by ACh(5.32 mM), DMPP(100 uM for 1 min), McN-A-343(200 uM for 2 min), Bay-K-8644(10 uM) and high potassium(56 mM), while it did not affect the CA secretion of cyclopiazonic acid(10 uM). Also, in the presence of thiopental-Na (100 uM), CA secretory responses evoked by ACh, DMPP, McN-A-343 and high K+ were markedly depressed. Moreover, in adrenal glands preloaded with ketamine(100 uM for 20 min), which is known to be a dissociative anesthetic, CA secretion evoked by ACh, DMPP, McN-A-343 and high K+ were significantly attenuated. CONCLUSION: Taken together, these experimental results suggest that pentobarbital-Na depresses CA release evoked by both cholinergic stimulation and membrane-depolarization from the isolated rat adrenal medulla and that this inhibitory activity may be due to the result of the direct inhibition of Ca++ influx into the chromaffin cells without any effect on the calcium mobilization from the intracellular store.