MiR-1-3p suppresses cell proliferation and invasion and targets STC2 in gastric cancer.

OBJECTIVE: MiR-1 has been reported to act as an inhibitory microRNA in gastric cancer (GC). This study aimed to investigate the regulatory mechanism by which miR-1-3p blocks the progression of GC by targeting stanniocalcin 2 (STC2). PATIENTS AND METHODS: The expression level of miR-1-3p in GC was assessed via quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Expressions of STC2 were measured by qRT-PCR and Western blot analysis. Proliferation and invasion assays were detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and transwell assays, respectively. Moreover, the dual-luciferase reporter assay was used to confirm the binding sites between miR-1-3p and STC2. RESULTS: MiR-1-3p was significantly down-regulated in GC. Moreover, abnormal expression of miR-1-3p was correlated with GC tumor size. Functionally, overexpression of miR-1-3p inhibited proliferation and invasion in GC by inhibiting stanniocalcin 2 (STC2) expressions. In contrast, STC2 was significantly up-regulated in GC. Furthermore, miR-1-3p negatively regulated STC2 expression in GC. The upregulation of STC2 weakened the inhibitory effect of miR-1-3p in GC. CONCLUSIONS: MiR-1-3p suppressed cell proliferation and invasion by targeting STC2 in GC, providing a novel therapeutic target for GC.

[The distribution and homology of mitochondrial plasmid-like DNAs in different cucumber varieties].

Four kinds of mitochondrial plasmid-like DNA, designated pC1, pC2, pC3 and pC4, have been found in cucumber (Jinyan No. 4). Their distribution in 14 Cucumber varieties was analyzed. Plasmid-like DNAs were detected in Jinchun No. 2, Jinchun No. 5, Jinxinmici, Jinlu No. 4 and Jinyan No. 4, and the rest 9 varieties contained no plasmid-like DNAs, suggesting that their distribution is irregular. There was homology among the same plasmid-like DNA family in different varieties. pC4 showed homology to the nuclear DNA of Jinyan No. 4, in addition to the nuclear genomes of other 7 varieties either with plasmid-like DNAs or without. The homologous sequences of pC4 in the cucumber nuclear DNA were repetitive. Sequences homologous to pC4 were also found in the nuclear genomes of towel gourd and pumpkin (other cucurbitaceous plant). Therefore, we propose that mitochondrial plasmid-like DNA occurred before cucumber diverged from cucurbit and had integrated into the nuclear DNA. The varieties without plasmid-like DNAs might lost them during evolution.

Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor.

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.

The histone deacetylase-3 complex contains nuclear receptor corepressors.

Acetylation and deacetylation of nucleosomal histones have profound effects on gene transcription in all eukaryotes. In humans, three highly homologous class I and four class II histone deacetylase (HDAC) enzymes have been identified to date. The class I deacetylases HDAC1 and HDAC2 are components of multisubunit complexes, one of which could associate with the nuclear hormone receptor corepressor, N-CoR. N-CoR also interacts with class II deacetylases HDAC4, HDAC5, and HDAC7. In comparison with HDAC1 and HDAC2, HDAC3 remains relatively uncharacterized, and very few proteins have been shown to interact with HDAC3. Using an affinity purification approach, we isolated an enzymatically active HDAC3 complex that contained members of the nuclear receptor corepressor family. Deletion analysis of N-CoR revealed that HDAC3 binds multiple N-CoR regions in vitro and that all of these regions are required for maximal binding in vivo. The N-CoR domains that interact with HDAC3 are distinct from those that bind other HDACs. Transient overexpression of HDAC3 and microinjection of Abs against HDAC3 showed that a component of transcriptional repression mediated by N-CoR depends on HDAC3. Interestingly, data suggest that interaction with a region of N-CoR augments the deacetylase activity of HDAC3. These results provide a possible molecular mechanism for HDAC3 regulation and argue that N-CoR is a platform in which distinct domains can interact with most of the known HDACs.

[Properties and nucleotide sequence of mitochondrial plasmid-like DNA pC1 of cucumber].

Four kinds of plasmid-like DNA, designated pC1, pC2, pC3 and pC4 were found in the mitochondrion of cucumber Jinyan No. 4. The circle plasmid-like DNA pC1 was cloned into the EcoR I site of pUC19 using E. coli JM109 as host. The cloned pC1 DNA was isolated and used as probes in Southern analyses of total mitochondrial DNA, nuclear DNA and chloroplast DNA. Evidences were obtained that the pC1 did not show any homology with nuclear, chloroplast, main mitochondrial genomes and the other plasmid-like DNAs. Sequence analysis revealed that pC1 was 2,889 bp long. It contained many forward and reverse repeat sequences. Three main open reading frames in pC1 were longer than 800 bp. Computer-assisted searching the nucleotide sequence in GenBank database revealed pC1 had no significant homology with known sequences of mitochondrial and plasmid-like DNA, but had homology with the E. coli, Mycobaterium tuberculosis and Anacystis nidulans genomes. The predicted proteins of pC1 main ORFs show homology with the sulfate transport system in bacteria, alga and liverwort. It suggested that pC1 may encode functional proteins.

Identification and characterization of a stress-responsive promoter in the macromolecular synthesis operon of Bacillus subtilis.

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant. Induction of sigmaA has been observed exclusively in this mutant harbouring extra copies of the plasmid-borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B. subtilis macromolecular synthesis (MMS; rpoD or sigA) operon. Investigation of the mechanisms leading to the induction has allowed us to identify a sigmaB-type promoter, P7, in the MMS operon for the first time. Therefore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the four known sigmaA- and sigmaH-type promoters, as well as two incompletely defined promoters. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress. The significant heat induction of P7 in B. subtilis DB1005 harbouring a plasmid-borne Ts sigA gene can be explained by a model of competition between sigmaA and sigmaB for core binding; very probably, the sigmaB factor binds more efficiently to core RNA polymerase under heat shock. This mechanism may provide a means for the expression of the B. subtilis MMS operon when sigmaA becomes defective in core binding.

The importance of a proper helical structure in the promoter-10 binding region to Bacillus subtilis sigma A structure and function.

Two Bacillus subtilis sigA mutants with amino acid substitutions tending to disrupt the structure of the promoter -10 binding helix of B. subtilis sigma A factor were constructed. B. subtilis DB1001 which contained an A197P substitution was very sensitive to temperature elevation. B. subtilis DB1002 had a T199G substitution and was low in growth potential at the elevated temperature. Degradation of sigma A in B. subtilis DB1001 (t(1/2)=63.2 min) and DB1002 (t(1/2)=186.0 min) occurred readily even at 37 degrees C; however, sigma A in B. subtilis DB2 (wild-type) was fairly stable at the same temperature. The activities of both DB1001 and DB1002 sigma A factors on groE promoter (sigma A-type) were lower than those of the wild-type counterpart at both permissive and restrictive temperatures. The failure of a higher sigma A concentration to suppress the Ts phenotype of DB1001 indicated that the temperature sensitivity of B. subtilis DB1001 was due to altered function, rather than insufficient concentration, of sigma A in the cells. Taken together, our results suggest that the helicity of the promoter -10 binding helix is essential to the packing interaction in the hydrophobic core region of sigma A, which helps to maintain the stable and functional sigma A structure.

Comparative brain exposure to (-)-carbovir after (-)-carbovir or (-)-6-aminocarbovir intravenous infusion in rats.

PURPOSE: Evaluate the ability of (-)-6-aminocarbovir ((-)-6AC) to improve the CNS exposure to (-)-carbovir ((-)-CBV). METHODS: Activation of (-)-6AC in vitro was assessed by incubations of rat brain tissue homogenates. The in vivo brain exposure to (-)-CBV was then examined in rats after iv infusions of either (-)-CBV (n = 4) or (-)-6AC (n = 5). The drugs were infused to steady-state via the jugular vein. At the end of the infusion, a bolus of [3H]inulin was injected via the femoral vein in order to obtain an estimate of the brain vascular space. RESULTS: (-)-6AC was converted to (-)-CBV by incubations of rat brain tissue homogenates. After iv infusion of (-)-CBV, the brain/blood concentration ratio of (-)-CBV was 0.032 +/- 0.009. The brain/blood concentration ratio of (-)-CBV after iv infusion of (-)-6AC was 0.080 +/- 0.020. CONCLUSIONS: (-)-6AC improved the brain delivery of (-)-CBV, although the absolute exposure of the brain tissue to (-)-CBV was still quite low.

The response of a Bacillus subtilis temperature-sensitive sigA mutant to heat stress.

The mutant sigA allele of Bacillus subtilis DB1005 was confirmed to be temperature sensitive (ts) and transferable among strains of B. subtilis by chromosomal transformation and gene conversion. This ts sigA allele had a pleiotropic effect on gene expression of DB1005. The induction of certain heat shock proteins in DB1005 was markedly less significant than that observed in the wild-type strain (DB2) under heat stress. In contrast, some proteins required for coping with oxidative stress and glucose starvation were induced abruptly in DB1005 but not in DB2. Heat induction of the groEL gene in vivo at both transcription and translation levels was much lower in DB1005 than in DB2. Besides, the putative sigma A-type promoter from the groESL operon of B. subtilis was able to be transcribed by the reconstituted sigma A RNA polymerase in vitro at both 37 and 49 degrees C. These results strongly suggest that the expression of the groEL gene of B. subtilis under heat stress is regulated at least in part by sigma A at the level of transcription. Our results also showed that DB1005 did not respond too differently from the wild type to ethanol stress, except after a relatively long exposure.