circRERE regulates the expression of GBX2 through miR-1299 and ZC3H13/N6-methyladenosine (m6A) to promote growth and invasion of hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours in the world. Current studies have shown that circular RNAs (circRNAs) and N6-methyladenosine (m6A) methylation play important roles in the progression of HCC, but further studies are needed to confirm the underlying mechanisms. The expression of circRERE was significantly upregulated in HCC cells, and its downregulation reduced HCC cell viability and invasion while increasing apoptosis. Further study showed that circRERE bound directly to miR- 1299. After downregulating the expression of circRERE, miR-1299 expression was significantly enhanced, while the expression of its downstream target gene GBX2 was suppressed, indicating that circRERE promoted the expression of GBX2 through miR-1299. In addition, downregulation of circRERE expression significantly increased the m6A level of GBX2 and promoted the expression of methyltransferase ZC3H13, while overexpression of ZC3H13 significantly inhibited the expression of GBX2 but increased its m6A methylation. circRERE could regulate the m6A modification of GBX2 through ZC3H13, thus promoting the expression of GBX2.GBX2 was upregulated in HCC tissues, while miR-1299 and ZC3H13 were downregulated. MiR-1299 mimics, ZC3H13 overexpression or GBX2 siRNA significantly inhibited HCC cell viability, promoted apoptosis and reduced invasion; GBX2 exerted the opposite effects and could reverse the regulatory effects of miR-1299 or ZC3H13 on HCC cells. Therefore, circRERE promotes the growth and invasion of HCC cells by regulating the expression of GBX2 through miR-1299 and ZC3H13/m6A, indicating that it is a key circRNA in the progression of HCC.

Evaluation of the prognostic value of the metastatic lymph node ratio for gastric cancer.

BACKGROUND: The aim of this study was to investigate the prognostic value of metastatic lymph node (LN) ratio (LNR) compared with pathologic node (pN) category. METHODS: Three hundred ninety-nine patients with gastric cancer with R0 resection were reviewed. LNR, pN, and the number of retrieved LNs were evaluated in node-positive groups with ≥15 or <15 LNs resected and a node-negative group, respectively, by univariate and multivariate analyses. Associations of pN and LNR with the number of retrieved LNs were determined using Spearman's rank correlation test. RESULTS: LNR and pN were correlated with overall survival. For the node-positive group with ≥15 LNs retrieved, pN and LNR were independent prognostic factors, with the hazard ratio higher for LNR; neither was correlated with the number of retrieved LNs. For the group with <15 LNs retrieved, LNR but not pN was an independent prognostic factor, with LNR uncorrelated with the number of LNs retrieved. For the node-negative group, the number of LNs retrieved retained an independent prognostic factor. CONCLUSIONS: LNR is an independent prognostic factor in node-positive patients with gastric cancer with R0 resection, and it is uninfluenced by the number of LNs retrieved. It may be superior to pN.

[Expression of alpha-tubulin and MDR1 and their correlation with the biological features of non-small cell lung carcinoma].

OBJECTIVE: To detect the expression of alpha-tubulin and MDR1 in human non-small cell lung carcinoma (NSCLC), and to clarify their clinical significance. METHODS: Paraffin embedded tissues from 158 primary non-small lung carcinomas and 30 paracancerous lung tissues were examined for expression of alpha-tubulin and MDR1 by immunohistochemistry (SP method). 30 freshly taken NSCLC tissues were examined by Western blot analysis. The relationship between alpha-tubulin and MDR1 expression and the biological features of lung carcinoma was analyzed. RESULTS: The positive rate of alpha-tubulin and MDR1 expressions in the lung carcinomas was 65.2% and 51.3%, respectively. There was no expression of either of them in 30 paracancerous lung tissues. Western blot analysis showed that the level of alpha-tubulin and MDR1 expressions in NSCLC tissues were 0.49 +/- 0.06 and 0.56 +/- 0.04, respectively, significantly higher than that in paracancerous tissues (0.07 +/- 0.01) (t = 3.693 and t = 6.769, P < 0.01). The positive rate of alpha-tubulin expression was gradually increased with tumor progression, significantly higher in III-IV stage cancers and in poorly differentiated carcinomas (both P < 0.01). There was a distinct statistically significant difference between stage I, stage II and III, and stage IV. The positive rate of alpha-tubulin in well-moderately differentiated carcinomas was lower than that in poorly differentiated ones. There was no significant correlation with age, sex, tumor size, histological type, clinical TNM system and lymph node metastasis. The positive rate of MDR1 was not correlated with sex, age, tumor size, pathological grading, clinical TNM stages and lymph node metastasis. But the positive rate of MDR1 in adenocarcinoma was significantly higher than that in squamous carcinoma and undifferentiated large cell carcinomas (P < 0.01). alpha-tubulin and MDR1 expression had no impact on the outcome of chemotherapy (chi(2) = 0.69 and 1.30, P > 0.05, respectively). Univariate analysis showed that the 5-year survival rate of patients with negative alpha-tubulin and MDR1 expression was 30.7% and 28.5%, respectively, significantly higher than that of patients with positive alpha-tubulin and MDR1 expression (13.5% and 11.8%, respectively) (chi(2) = 20.69 and 15.52, P < 0.01, respectively), and multivariate Cox regression analysis showed that alpha-tubulin (RR = 3.287, P = 0.006) and clinical TNM stage (RR = 1.954, P = 0.025) were significantly independent predictive factor for patients with lung cancer, MDR1 and other factors could not be used as an independent predicitive factors. However, there was no significant correlation between the expression of alpha-tubulin and MDR1 in lung carcinoma(r = 0.093, P > 0.05). CONCLUSION: The expression of alpha-tubulin and MDR1 may play an important role in the development and progression of human non-small cell lung carcinoma. Combined detection could be considered as an important index for predicting prognosis of lung carcinoma.

Phenoxypropylamines: synthesis and antiulcer evaluation.

We have synthesized a number of phenoxypropylamines from N-{3-[3-(1-piperidinylmethyl)phenoxy]propyl}chloroacetamide (3). All the products have been characterized by elemental analysis, (1)H-NMR and MS. The biological activity effects of the title compounds were examined. From the biological activity results, we found that two of themshowed significant gastric acid antisecretory activity.

Dominant-negative behavior of mammalian Vps35 in yeast requires a conserved PRLYL motif involved in retromer assembly.

The retromer protein complex assists in recycling selected integral membrane proteins from endosomes to the trans Golgi network. One protein subcomplex (Vps35p, Vps26p and Vps29p) combines with a second (Vps17p and Vps5p) to form a coat involved in sorting and budding of endosomal vesicles. Yeast Vps35p (yVps35) exhibits similarity to human Vps35 (hVps35), especially in a completely conserved PRLYL motif contained within an amino-terminal domain. Companion studies indicate that an R(98)W mutation in yVps35 causes defective retromer assembly in Saccharomyces cerevisiae. Herein, we find that the expression of hVps35 in yeast confers dominant-negative vacuolar proenzyme secretion and defective secretory proprotein processing. The mutant phenotype appears to be driven by hVps35 competing with endogenous yVps35, becoming incorporated into defective retromer complexes and causing proteasomal degradation of endogenous Vps26 and Vps29. Increased expression of yVps35 displaces some hVps35 to a 100 000 x g supernatant and suppresses the dominant-negative phenotype. Remarkably, mutation of the conserved R(107)W of hVps35 displaces some of the protein to the 100 000 x g supernatant, slows protein turnover and restores stability of Vps26p and Vps29p and completely abrogates dominant-negative trafficking behavior. We show that hVps35 coprecipitates Vps26, whereas the R(107)W mutant does not. In pancreatic beta cells, the R(107)W mutant shifts hVps35 from peripheral endosomes to a juxtanuclear compartment, affecting both mannose phosphate receptors and insulin. These data underscore importance of the Vps35 PRLYL motif in retromer subcomplex interactions and function.

Structural features of vps35p involved in interaction with other subunits of the retromer complex.

The penta-subunit retromer complex of yeast mediates selective retrieval of membrane proteins from the prevacuolar endosome to the trans Golgi network. In this study, we set out to generate a panel of vps35 dominant-negative mutants that disrupt retromer-mediated cargo sorting. Mapping of the mutations revealed two types of alterations leading to dominant-negative behavior of the 944-amino acid protein: (i) mutations at or near the R(98) residue or (ii) C-terminal truncations exemplified by a nonsense mutation at codon 733. Both could be suppressed by overexpression of wild-type Vps35p, suggesting that these dominant-negative mutants compete for interactions with other retromer subunits. Interestingly, Vps35-R(98)W expression destabilized Vps26p while having no effect on Vps29p stability, while Vps35-Q(733)* expression affected Vps29p stability but had no effect on Vps26p. Measurement of Vps35/Vps26 and Vps35/Vps29 pairwise associations by coimmunoprecipitation in the presence or absence of other retromer subunits indicated that the R(98) residue, which is part of a conserved PRLYL motif, is critical for Vps35p binding to Vps26p, while both R(98) and residues 733-944 are needed for efficient binding to Vps29p.

Effect of sulfonic acid containing mesogens on liquid-crystalline behavior of polysiloxane-based polymers.

A series of liquid-crystalline polysiloxanes synthesized by cholest-5-en-3-ol (3beta)-10-undecenoate and 4'-octanoyloxy-biphenyl-4-yl 4-allyloxy-3-sulfo-benzoate were prepared in a one-step reaction with sulfonic acid group contents ranging between 0 and 2.73 wt %. All the polymers displayed smectic mesophases with a large temperature range for the mesophases. With an increase of sulfonic acid containing mesogens in the polymers, the temperature of the glass transition did not change greatly, while the temperature of the clear point decreased. The hydrogen-bonding mesogen aggregates in the domains disturb the liquid-crystalline molecular mobility and orientation, leading to a decrease in temperature from the mesophase to the isotropic transition. Unlike the polymers containing lower sulfonic acid mesogens, some polymers showed a dendritic texture of the SmB* phase, indicating that the sulfonic mesogens enhanced the rigid moieties of the supermolecular structure of the liquid-crystalline phases. All the polymers displayed sharp and strong peaks at low angles around 2theta approximately 2.6 degrees and broad peaks at wide angles around 2theta approximately 17 degrees in X-ray measurements. The intensity of the strong peak at low angles in the X-ray profiles decreased with an increase of sulfonic acid mesogens in the polymer systems.

Optical characterization of polymer liquid crystal cell exhibiting polymer blue phases.

The optical properties of polymer liquid crystal cell exhibiting polymer blue phases (PBPs) have been determined using ultraviolet-visible spectrophotometry, polarizing optical microscopy (POM), differential scanning calorimetry (DSC), X-ray measurements, FTIR imaging and optical rotation technique. PBPs are thermodynamically stabile mesophases, which appear in chiral systems between isotropic and liquid crystal phases. A series of cyclosiloxane-based blue phase polymers were synthesized using a cholesteric LC monomer and a nematic LC monomer, and some of the polymers exhibit PBPs in temperature range over 300 degrees in cooling cycles. The unique property based on their structure and different twists formed and expect to open up new photonic application and enrich polymer blue phase contents and theory.

[Expression of epithelial-cadherin, CD44v6 and connexin43 in hepatocellular carcinoma].

OBJECTIVE: To study the expression of epithelial-cadherin (E-cad), CD44v6 and Cx43 in hepatocellular carcinoma (HCC) and its relationship with sex and age of patients, as well as tumor histopathologic grades. METHODS: Double immunofluorescent staining and laser scanning confocal microscopy was used to study the expression of E-cad, CD44v6 and Cx43 in 30 cases of normal liver tissue, 25 cases of benign hepatic lesions and 38 cases of HCC. In the HCC group, correlation of antigen expression with sex and age of patients and tumor histopathologic grades was studied by T-test. RESULTS: Significant decrease in expression of E-cad and Cx43 was noted in HCC group, as compared to normal liver tissue and benign hepatic lesion (P<0.05). On the other hand, CD44v6 expression was higher in HCC group than in the other two groups (P<0.05). In HCC group, the expression of E-cad and Cx43 did not correlate with sex, age and histopathologic grades (P>0.05). However, CD44v6 expression positively correlated with higher tumor histopathologic grades (P<0.05) but not sex and age of patients (P>0.05). In HCC group, the expression of E-cad positively correlated with that of Cx43, while the expression of CD44v6 negatively correlated with that of E-cad and Cx43. CONCLUSIONS: Tumor immunophenotype alters during development and progression of HCC. Low expression of E-cad and Cx43 and high expression of CD44v6 may be related to aggressive clinical behavior of HCC, moreover, high expression of CD44v6 correlated with high tumor grades. Detection of E-cad, CD44v6 and Cx43 expression may thus be useful in predicting prognosis of HCC.

Behavior in the eukaryotic secretory pathway of insulin-containing fusion proteins and single-chain insulins bearing various B-chain mutations.

In the secretory pathway, endoproteolytic cleavage of the insulin precursor protein promotes a change in the biophysical properties of the processed insulin product, and this may be relevant for its intracellular trafficking. We have now studied several independent point mutants contained within the insulin B-chain, S9D, H10D, V12E (called B9D, B10D, and B12E), as well as the double point mutant P28K,K29P (B28K,B29P), that have been reported to inhibit insulin oligomerization. In yeast cells, the unprocessed precursor of each of these mutants is secreted, whereas >90% of the endoproteolytically released single-chain insulin moiety is retained intracellularly; a large portion of the B9D, B10D, and B12E single-chain insulins exhibit abnormally slow mobility upon nonreducing SDS-PAGE, despite normal mobility upon reducing SDS-PAGE. Although no free thiols can be detected, each of these mutants exhibits increased disulfide accessibility to dithiothreitol. After dithiothreitol treatment, a portion of the molecules can reoxidize to a form more compact than the original single-chain insulin mutants formed in vivo (indicating initial disulfide mispairing). Disulfide mispairing of a fraction of B9D, B10D, and B12E mutants also occurs in the context of single-chain insulin and even in authentic proinsulin expressed within the secretory pathway of mammalian cells. We conclude that analyses of the intracellular trafficking of certain oligomerization-defective insulin mutants is complicated by the formation of disulfide isomers in the secretory pathway.

Lumenal protein multimerization in the distal secretory pathway/secretory granules.

Differences in protein solubility appear to play an important role in lumenal protein trafficking through Golgi/post-Golgi compartments. Recent advances indicate that multimeric protein assembly is one of the factors regulating the efficiency of protein storage within secretory granules, by mechanisms that, with slight modification, might be considered to represent the culmination of a process of Golgi cisternal maturation.

A convenient method for the synthesis of amine-terminated poly(ethylene oxide) and poly(epsilon-caprolactone).

A convenient synthetic route to prepare amine-terminated poly(ethylene oxide) (PEO) and poly(epsilon-caprolactone) (PCL) was described. The strategy involved two-step reactions, the condensation of hydroxyl-terminated PEO and PCL with N-benzyloxycarbonyl amino acid followed by the catalytic hydrogenation under mild conditions. NMR and GPC measurements indicated that the reactions proceeded nearly quantitatively. Amine-terminated PEO thus prepared was used to initiate the polymerization of alpha-(N(epsilon)-benzyloxycarbonyl-L-lysine) N-carboxy anhydride [lys(Z)-NCA], and the results confirmed that the reactivity of the amino group was high.

Secretory Expression of Porcine Insulin Precursor in Kluyveromyces lactis and Its Conversion into Human Insulin.

Porcine insulin precursor (PIP) was cloned to vectors derived from plasmid pKD1 and expressed in Kluyveromyces lactis. The secretory expression level of PIP was 20 to 30 mg per liter of the culture medium. Human insulin obtained from PIP through tryptic transpeptidation was characterized. Its amino acid composition, crystalline shape and biological activity are identical with those of native insulin.