miR-424 protects PC-12 cells from OGD-induced injury by negatively regulating MKP-1.

OBJECTIVE: It's of great significance to investigate the novel targets of drugs for the treatment of stroke. In this study, we explored the neuroprotective role of miR-424 in oxygen glucose deprivation (OGD)-induced injuries in PC-12 cells. MATERIALS AND METHODS: PC-12 cells were subjected to OGD stimulation to mimic ischemic injury. The expressions of miR-424 and mitogen-activated protein kinase phosphatase-1 (MKP-1) were altered by transient transfection with miR-424 mimic, miR-424 inhibitor, pEX-MKP-1, or sh-MKP-1. Cell counting kit-8 (CCK-8) assay, flow cytometry, and quantitative reverse transcription polymerase chain reaction (qRT-PCR), were conducted to respectively detect cell viability, apoptotic cells, and the expression of miR-424 and MKP-1. The protein expressions of several factors were determined by Western blot. Meanwhile, relative luciferase activity assay was done to verify the predicted targets association. RESULTS: OGD induced injury in PC-12 cells by suppressing cell viability and inducing apoptosis. OGD also induced the expression of miR-424 in PC-12 cells. Overexpression of miR-424 protected PC-12 cells from OGD-induced injury by increasing cell viability and decreasing apoptosis. MKP-1 was a direct target of miR-424, and its expression was negatively regulated by miR-424. Up-regulation of expression of MKP-1 aggravated OGD-induced cell injury by inhibiting the expression of hypoxia-inducible factor 1α (HIF-1α), thus inhibiting the PI3K/AKT/mTOR pathways. CONCLUSIONS: miR-424 protected PC-12 cells from OGD-induced injury through direct suppression of MKP-1 expression, as MKP-1 promoted OGD-induced cell injury by inhibiting the expression of HIF-1α and PI3K/AKT/mTOR pathways.

Expression of long non-coding RNA CRNDE in glioma and its correlation with tumor progression and patient survival.

OBJECTIVE: Long non-coding RNAs (lncRNAs) CRNDE has been identified as a tumor oncogene in glioma. However, its clinical significance and prognostic value in glioma have not been investigated until now. The aim of this study was to explore CRNDE expression levels and evaluated its clinical significance in glioma patients. PATIENTS AND METHODS: Expression levels of lncRNA CRNDE in 164 glioma specimens were determined by quantitative real-time PCR (qRT-PCR). The chi-square test was used to explore CRNDE expression with respect to clinicopathological parameters. The overall survival was analyzed by log-rank test, and survival curves were plotted according to Kaplan-Meier. Univariate and multivariate analyses were performed to analyze the prognostic significance of CRNDE expression. RESULTS: Compared with nonneoplastic brain tissues, the expression level of CRNDE was significantly increased in glioma tissues (p < 0.01). CRNDE upregulation was correlated with larger tumor size (p = 0.011), higher WHO grade (p = 0.001), and recurrence (p = 0.008). Also, survival analysis proved that up-regulated CRNDE expression was associated with poor overall survival of glioma patients (p < 0.001). The multivariate Cox regression analysis indicated that CRNDE expression was an independent prognostic factor for overall survival. CONCLUSIONS: These results indicated that lncRNA CRNDE was associated with tumor progression and could be an independent prognostic factor for glioma patients.

A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy et al. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133 nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.

Quantitative assessment of the association between MTHFR C677T polymorphism and colorectal cancer risk in East Asians.

A great number of studies regarding the association between MTHFR C677T polymorphism and risk of colorectal cancer (CRC) in East Asians were published, but the results were inconsistent. Thus, a meta-analysis was performed to investigate the association. PubMed, Embase, and CBM databases were searched for eligible publications. Pooled odds ratios (ORs) with 95 % confidence intervals (95 % CIs) were calculated using random or fixed effect models. Finally, 24 case-control studies with a total of 7,230 CRC cases and 9,285 controls were included. Meta-analyses of a total of 24 studies showed there was a statistically significant association between MTHFR C677T polymorphism and decreased CRC risk in East Asians under four genetic models (T versus C, OR = 0.92, 95 % CI 0.85-0.99; TT versus CC, OR = 0.80, 95 % CI 0.69-0.94; TT versus CT/CC, OR = 0.82, 95 % CI 0.71-0.95; TT/CT versus CC, OR = 0.92, 95 % CI 0.86-0.98). The cumulative meta-analyses for the allele contrast (T versus C), homozygote (TT versus CC), dominant (TT/CT versus CC), and recessive (TT versus CT/CC) models all showed a trend of more obvious association as information accumulated by year. Subgroup analyses by country further identified this association in Korea and Japan. This meta-analysis suggests that MTHFR C677T polymorphism is associated with decreased risk of colorectal cancer in East Asians, and MTHFR 677T variant has a protective effect on colorectal cancer.

Function analysis of a new type I PKS-SAT domain by SAT-EAT domain replacement.

The function of a new starter unit acyltransferase (SAT) domain SAT-EF080951 (GenBank accession number) encoded in a new type I polyketide synthase (PKS) gene cluster EF568935 (GenBank accession number) isolated for this study was analyzed by domain replacement with an extender unit AT (EAT) domain of avermectin PKS. It was shown that the SAT-EF080951 incorporated malonyl-CoA specifically in vivo, which contradicted the specificity that we had previously determined by substrate binding test in vitro. The result of this study indicates that type I PKS-SAT can alter its specificity in vivo and functions well in extender units and proved the feasibility of the SAT-EAT domain replacement in type I PKS. We propose that SAT-EAT replacement strategy could be a novel route for increasing the diversity of new polyketides combinatorially biosynthesized. The new type I PKS-SAT-EF080951 studied herein may be further employed for related studies on enzymology or combinatorial biosynthesis of polyketides.

Macrolactin S, a novel macrolactin antibiotic from marine Bacillus sp.

A new 24-membered ring lactone, macrolactin S, was isolated from a culture broth of marine Bacillus sp. and its structure was established by various spectral analyses. Macrolactin S is the first macrolactin hydroxylated at C-12. Besides, the known compounds macrolactin A and macrolactin B were also isolated, which were elucidated on the basis of spectral data analyses and literature data comparison. They exhibited antibacterial activity against E. coli and S. aureus.

Expression of estrogen receptor (ER) -alpha and -beta transcripts in the neonatal and adult rat cerebral cortex, cerebellum, and olfactory bulb.

In the present study expression of estrogen receptor subtype -alpha (ERalpha) and -beta (ERbeta) in the cerebral cortex, cerebellum, and olfactory bulb was investigated and compared between neonatal (1 to approximately 3-days-old) and adult (250 to approximately 350 g) rats, using reverse transcription-polymerase chain reaction (RT-PCR). No ERalpha transcripts were detectable in the adult cerebellum and olfactory bulb, whereas very weak expression of ERalpha was present in the adult cerebral cortex. No significant difference in ERbeta transcripts was detectable between the neonatal and adult rats. While transcripts for both ER subtypes were co-expressed in these brain areas of neonatal rats, although ERalpha expression was significantly weaker than ERbeta. Even in the cerebral cortex known to contain both ER subtypes in adult rats, ERalpha transcripts in neonatal rats were much higher than in adult. These observations provide evidence for the existence of different expression patterns of ERalpha/ERbeta transcripts in these three brain areas between the neonatal and adult rats, suggesting that each ER subtype may play a distinct role in the regulation of differentiation, development, and functions of the brain by estrogen.

[Effect of scald on gene transcription and content of endothelin-1 in supraoptic nucleus of rat hypothalamus].

Endothelin-1 (ET-1) gene transcription and endothelin-1-immunoreactivity (ET-1-ir) in the supraoptic nucleus (SON) of rat hypothalamus were respectively observed by in situ hybridization and immunohistochemistry after scald. Intensity of ET-1 mRNA and endothelin-1-immunoreactivity (ET-1-ir) was quantified by image analysis. Compared with the control (sham scald) group, no significant change in the intensity of ET-1 mRNA positive hybridization signals in SON was found 15 min post-scald, while there was a 35.1% increase in the positive hybridization signal intensity 60 min post-scald (P<0.05) and a 62.4% increase 180 min post-scald (P<0.01). The content of ET-1-ir in SON decreased significantly to 8.5% of the control 15 min post-scald (P<0.01), and gradually recovered to 31.5% and 52.4% of the control 60 min and 180 min post-scald respectively, though still significantly lower than the control (P<0.01). Pre- and post-scald ET-1 gene transcription in rat hypothalamus was also measured by Northern blot hybridization. No significant difference in the quantity of ET-1 mRNA was found between 15 min post-scald data and those of the control. The quantity increased to a significantly higher level 60 min post-scald (P<0.05) and further increased to 2.5 creased to 2.5 fold of the control 180 min post-scald (P<0.05). In addition, the Northern blot hybridization showed that the post-scald size of ET-1 mRNA remained unchanged despite of the increase in quantity. In view of the neuroendocrine role of SON, the changes in ET-1 mRNA and ET-1-ir in SON resulting from scald suggest that ET-1 may play an important role in neuroendocrine reactions following scald.

[The chemical degradation of lipopolysaccharides from oral anaerobes]

OBJECTIVE:To evaluate the effect of drug on chemical degradation of lipopolysaccharide (LPS) from oral anaerobes.METHODS:LPSs from porphyromonas gingivalis (Pg),bacteroides fragilis (Bf) and fusobacteria nucleatum (Fn) were extracted by the hot phenol-water method and purified by the phenol-chloroform-petroleum ether procedure.1.0 ml (200microg) various LPSs were incubated with 2.0 ml various drugs at 37degrees centigrade for 15 or 30 minutes in vitro,respectively.The chemical degradation of LPS was quantitated by limulus synthetic chromogenic substrated method after dialysis.RESULTS:The order of degradation was 30% hydrogen peroxide (H),50% citric acid (C),garlic guice (G),1:1 diluted G,25% C and 3% H,and their efffcts were dose dependent and were time dependent except H but the effect of lysozyme was minimal.CONCLUSION:The study may imply that the dose and the mechanism of various drugs on LPS degradation are different and remains to be elucidated.

[Chemical analysis of lipopolysac-charides derived from oral anacerobes]

Lipopolysaccharides were isolated from six oral anaerobes(Va.Bg.Bf.Fn.Aa.Co.)by the hot phenol-water procedure and purified by PCP method.The six species can be all extracted their LPS with different yields (from 0.70 to 2.83% of cell dried weight).Comparable of LPS with the enterobacteriaceae(Sm.),they were the constructed with hexosamine,fatty acids and phosphorus etc.On the other hand,they showed much distinct differences from the enterobacterial LPS structure as follow:(1) 2-keto-3-deoxy-D-mannooctonate(KDO) was not detected by routine procedure.(2)The amount of heptose is reltively lower.(3) Methylated and branched fatty acids were detected.These chemical characteristics may relate to biological activities and pathogenic potential of LPS and bacteria themselves.

Characterization of the lipid A component of genuine smooth-form lipopolysaccharide.

The smooth-form lipopolysaccharide of Salmonella abortus equi had earlier been separated into three distinct fractions, a long-chain fraction with an O chain containing 20-50 repeating units, a short-chain fraction consisting of an R lipopolysaccharide and another with 1-6 repeating units, and an R fraction identical to the lipopolysaccharide synthesized by Ra.b-mutant bacteria [Galanos et al. (1988) J. Chromatogr. 440, 397-404]. In this paper, the corresponding lipid A from each fraction was prepared by a newly elaborated procedure based on hydrolysis of the fractions in calcium acetate buffer (pH 3.5) followed by separation of the resulting free lipid A from the polysaccharide on a Sephadex G-100 column. Chemical analysis revealed that lipid A of the R fraction contained the expected spectrum and amounts of fatty acids and it proved to be structurally identical to lipid A of previously studied Salmonella R mutants. In contrast, the lipid A of the long-chain fraction contained only about 60% fatty acids compared to that of the R fraction. The lipid A of the short-chain fraction also expressed a reduced substitution pattern of acyl residues.

Large-scale fractionation of S-form lipopolysaccharide from Salmonella abortus equi. Chemical and serological characterization of the fractions.

The S-form lipopolysaccharide of Salmonella abortus equi was separated by a newly elaborated extraction method with organic solvents into three fractions of different chain length of the O-polysaccharide they contained. The three fractions were designated long-chain (20-50 repeating units), short-chain (0-6) and R-fraction (no repeating units) according to their migration pattern in polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. The nature of the fractions as long- and short-chain and as R-fraction was confirmed by chemical analysis. The concentration of O-specific sugars was highest in the long-chain fraction, where their molar ratio to glucosamine was ca. 25:1. In the short-chain fraction the ratio of O-sugars to glucosamine was 2.5:1, and in the R-fraction O-specific sugars were absent. The serological properties of the three fractions were in good agreement with their chemical composition.