Concentration-dependent mitogenic and antiproliferative actions of 2-methoxyestradiol in estrogen receptor-positive human breast cancer cells

We compared in this study the effects of 2-methoxyestradiol (2-MeO-E(2)) on the growth of two estrogen receptor (ER)-negative human breast cancer cell lines (MDA-MB-231 and MDA-MB-435s) and two ER-positive human breast cancer cell lines (MCF-7 and T-47D). 2-MeO-E(2) exerted a concentration-dependent antiproliferative action in the ER-negative MDA-MB-231 and MDA-MB-435s cells. The presence or absence of exogenous 17beta-estradiol (E(2)) in the culture medium did not affect the potency and efficacy of 2-MeO-E(2)'s antiproliferative action in these ER-negative cells. When the ER-positive MCF-7 and T-47D cells were cultured in a medium supplemented with 10nM of exogenous E(2), 2-MeO-E(2) at 750 nM to 2 microM concentrations exerted a similar antiproliferative effect. However, when the ER-positive cell lines were cultured in the absence of exogenous E(2), 2-MeO-E(2) at relatively low concentrations (10-750 nM) had a moderate mitogenic effect, with its apparent efficacy 75-80% of that of E(2). This mitogenic effect of 2-MeO-E(2) was ER-mediated and largely attributable to 2-MeO-E(2)'s residual estrogenic activity on the basis of our following findings: (i) its effect was only manifested in the ER-positive cells but not in the ER-negative cells; (ii) its effect in the ER-positive cells was partially or fully abolished when exogenous E(2) was concomitantly present in the culture medium; (iii) 2-MeO-E(2) retained 1-2% of E(2)'s binding affinity for the human ERalpha and ERbeta, and its mitogenic effect was inhibited in a concentration-dependent manner by ICI-182,780, a pure ER antagonist; and (iv) its effect was not due to its metabolic conversion to 2-hydroxyestradiol. Our timely findings are of importance to the on-going clinical trials designed to evaluate 2-MeO-E(2)'s effectiveness for the treatment of different types (ER-positive or ER-negative) of human breast cancer. This knowledge will improve the design of clinical trials as well as the interpretation of clinical outcomes when 2-MeO-E(2) is used as a single agent therapy or as part of a combination therapy for human breast cancer.     

Identification and Functional Analysis of dTDP-Glucose-4,6-Dehydratase Gene and Its Linked Gene Cluster in an Aminoglycoside Antibiotics Producer of <Emphasis Type="Italic">Streptomyces tenebrarius</Emphasis> H6

Streptomyces tenebrarius H6 produces a variety of aminoglycoside antibiotics, such as apramycin, tobramycin, and kanamycin B. Primers were designed according to the highly conserved sequences of the dTDP-glucose-4,6-dehydratase genes, and a 0.6-kb PCR product was obtained from S. tenebrarius H6 genomic DNA. With the 0.6-kb PCR product as a probe, a BamHI 7.0-kb fragment was isolated. DNA sequence analysis of the 7.0-kb fragment revealed four ORFs and an incomplete ORF. In search of databases, the deduced product of one ORF (orfE) showed 62% identity to the dTDP-glucose-4,6-dehydratase, StrE of S. griseus. Three other ORFs (orfG1, orfG2, and orfGM) showed 55%, 62%, and 42% similarities, respectively, to glycosyltransferase from Clostridium acetobutylicum and mannosyltransferase from Xanthomonas axonopodis pv. citri str. 306 and glycosyltransferase from Pseudomonas putida KT2440. Upstream of the orfE was an incomplete ORF, and the deduced product showed 56% similarity to dTDP-4-dehydrorhamnose, StrL from S. griseus. The function of the orfE gene was studied by targeted gene disruption. The resulting mutant failed to produce tobramycin and kanamycin B, but still produced apramycin, suggesting that the orfE gene and linked gene cluster are essential for the biosynthesis of tobramycin and kanamycin B in S. tenebrarius H6.     

慢性间歇性低氧降低急性缺氧对大鼠肺动脉平滑肌细胞膜上电压门控性钾通道的抑制

为了从离子通道水平上探讨机体低氧适应的离子机制,本实验将雄性 SD 大鼠随机分为常氧对照组和慢性间歇性低氧组[氧浓度(10 ± 0.5) %, 间断缺氧每天 8 h]。用酶解法急性分离单个大鼠肺内动脉平滑肌细胞(pulmonary artery smoothmuscle cells, PASMCs),以全细胞膜片钳技术记录 PASMCs 膜上的电压门控性钾通道 (voltage-gated potassium channel, KV) 电流,观察急性缺氧对慢性间歇性低氧大鼠 PASMCs 的 KV 的影响, 为机体适应低氧能力提供实验依据。结果显示:⑴常氧对照组在电流钳下,急性缺氧可使膜电位明显去极化(由-47.2 ±2.6 mV 去极到 -26.7 ±1.2 mV ); 在电压钳下, 急性缺氧可显著抑制 KV电流( 60 mV 时, KV电流密度从 153.4 ± 9.5 pA/pF降到 70.1 ± 10.6 pA/pF), 峰电流的抑制率为(57.6 ± 3.3) %, 电流-电压关系曲线向右下移。⑵慢性间歇性低氧组KV电流密度随低氧时间延长而逐渐减少(慢性低氧10 d后就有显著性意义),电流- 电压关系曲线逐渐右下移。⑶急性缺氧对慢性间歇性低氧大鼠PASMCs KV电流的抑制作用随慢性间歇性低氧时间延长而逐渐减弱。上述观察结果提示慢性间歇性低氧减弱急性缺氧对 KV 的抑制, 这可能是机体低氧适应的一种重要机制。     

Expression and characteristics of the gene encoding azoreductase from Rhodobacter sphaeroides AS1.1737

A gene that encodes a protein with azoreductase activity was obtained by PCR amplification from Rhodobacter sphaeroides AS1.1737. The enzyme, with a molecular weight of 18.7 kD, was heterologously expressed in Escherichia coli and its azoreductase activity was characterized. Furthermore, the reduction mechanism of azo dyes catalyzed by the azoreductase was studied in detail. The presence of a hydrazo-intermediate was identified, which provided a convincing evidence for the assumption that azo dyes were degraded via an incomplete reduction stage.     

Cloning and over-expression of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.     

Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains. Genes encoding many of these cysteine-rich RLKs (CRKs) are induced by pathogen infection, suggesting a possible role in plant defense responses. We have previously generated Arabidopsis plants expressing four pathogen-regulated CRK genes (CRK5, 6, 10 and 11) under control of a steroid-inducible promoter and found that induced expression of CRK5, but not the other three CRK genes, triggered hypersensitive response-like cell death in transgenic plants. In the present study, we have analyzed the structural relationship of the CRK family and identified three CRKs (CRK4, 19 and 20) that are structurally closely related to CRK5. Genes encoding these three CRKs are all induced by salicylic acid and pathogen infection. Furthermore, induced expression of CRK4, 19and 20 all activates rapid cell death in transgenic plants. Thus, the activity of inducing rapid cell death is shared by these structurally closely related CRKs. We have also performed yeast two-hybrid screens and identified proteins that interact with the kinase domains of CRKs. One of the identified CRK-interacting proteins is the kinase-associated type 2C protein phospohatase known to interact with a number of other RLKs through its kinase-interacting FHA domain. Other CRK-interacting proteins include a second protein with a FHA domain and another type 2C protein phosphatase. Interactions of CRKs with these three proteins in vivo were demonstrated through co-immunoprecipitation. These CRK-interacting proteins may play roles in the regulation and signaling of CRKs.     

Regulation of hypoxia-induced release of corticotropin-releasing factor in the rat hypothalamus by norepinephrine

Corticotropin-releasing factor (CRF) peptide release was activated by hypoxia in the rat hypothalamus. The mechanisms, however, of the hypoxia-induced CRF release remains unclear. In this study, we demonstrated that the norepinephrine (NE) and its receptors in the paraventricular nucleus (PVN) mediated the CRF release in a simulated altitude hypoxia. When rats were exposed to 5 or 7 km altitude of hypoxia for a short or long term: (1) NE levels in the PVN and the CeA, using the HPLC analysis, were intensity and time course dependently increased, but the increase in the PVN were potential than in the CeA. Restraint-induced NE increase was much higher in both the PVN and the CeA, compared with hypoxia-induced response. (2) Hypoxia and restraint significantly enhanced CRF release in the ME and the PVN but not in the CeA, through RIA assay, which result in stimulating corticosterone secretion. (3) Hypoxia-induced CRF release was reversed by an injection of prazosin (i.c.v.), an alpha-1 adrenoceptor antagonist, while administration of yohimbine (i.c.v.), an alpha-2 receptor antagonist, facilitated further CRF release. These data suggested that hypoxia induced NE activation centrally, via alpha-1 and -2 receptors, leading to improving hypothalamic CRF release, which in turn stimulated pituitary and adrenal cortex. Restraint presented much potential action on NE activation than hypoxia.     

ERIC-PCR fingerprinting-based community DNA hybridization to pinpoint genome-specific fragments as molecular markers to identify and track populations common to healthy human guts

Bacterial populations common to healthy human guts may play important roles in human health. A new strategy for discovering genomic sequences as markers for these bacteria was developed using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR fingerprinting. Structural features within microbial communities are compared with ERIC-PCR followed by DNA hybridization to identify genomic fragments shared by samples from healthy human individuals. ERIC-PCR profiles of fecal samples from 12 diseased or healthy human and piglet subjects demonstrated stable, unique banding patterns for each individual tested. Sequence homology of DNA fragments in bands of identical size was examined between samples by hybridization under high stringency conditions with DIG-labeled ERIC-PCR products derived from the fecal sample of one healthy child. Comparative analysis of the hybridization profiles with the original agarose fingerprints identified three predominant bands as signatures for populations associated with healthy human guts with sizes of 500, 800 and 1000 bp. Clone library profiling of the three bands produced 17 genome fragments, three of which showed high similarity only with regions of the Bacteroides thetaiotaomicron genome, while the remainder were orphan sequences. Association of these sequences with healthy guts was validated by sequence-selective PCR experiments, which showed that a single fragment was present in all 32 healthy humans and 13 healthy piglets tested. Two fragments were present in the healthy human group and in 18 children with non-infectious diarrhea but not in eight children with infectious diarrhea. Genome fragments identified with this novel strategy may be used as genome-specific markers for dynamic monitoring and sequence-guided isolation of functionally important bacterial populations in complex communities such as human gut microflora.     

The pharmacophore hypotheses of I(Kr) potassium channel blockers: novel class III antiarrhythmic agents

Predictive pharmacophore models were developed for a large series of I(Kr) potassium channel blockers as class III antiarrhythmic agents using HypoGen in Catalyst software. The pharmacophore hypotheses were generated using a training set consisting of 34 compounds carefully selected from documents. Their biological data, expressed as IC(50), spanned from 1.5 nM to 2.8 mM with 7 orders difference. The most predictive hypothesis (Hypo1), consisting of four features (one positive ionizable feature, two aromatic rings and one hydrophobic group), had a best correlation coefficient of 0.825, a lowest rms deviation of 1.612, and a highest cost difference (null cost-total cost) of 77.552, which represents a true correlation and a good predictivity. The hypothesis Hypo1 was then validated by a test set consisting of 21 compounds and by a cross-validation of 95% confidence level with randomizing the data using CatScramble program. Accordingly, our model has strong predictivity to identify structural diverse I(Kr) potassium channel blockers with desired biological activity by virtual screening