Low concentrations mono-butyl phthalate stimulates steroidogenesis by facilitating steroidogenic acute regulatory protein expression in mouse Leydig tumor cells (MLTC-1)

Di-n-butyl phthalate (DBP) is one of the most dominant phthalate esters and is widely distributed environmental contaminant. Although previous studies have demonstrated that DBP led to a variety of male reproductive abnormalities similar to those caused by androgen receptor antagonists, DBP and its active metabolite, mono-butyl phthalate (MBP), have been demonstrated no affinity for the androgen receptor, but rather exert anti-androgenic effect by altering testosterone biosynthesis. Furthermore, all these results were obtained from very high administrations of DBP or MBP. The purpose of this study was to determine the onset and the site of action of relatively low concentration of MBP on steroidogenesis in vitro. The mouse Leydig tumor cells (MLTC-1) was employed as a cellular model to investigate the effect of MBP on steroidogenesis. Various concentrations of MBP (1, 10, 100 and 1000nmol/l) and its solvent dimethyl sulfoxide (DMSO) were added to the medium for 24h followed by stimulation of some compounds such as human chorionic gonadotrophin (hCG), cholera toxin (CT), forskolin, cAMP analog 8-Br-cAMP, 22(R)-hydroxycholesterol (22R-HC) and pregnenolone. Progesterone in the medium and amounts of intracellular cAMP were measured by RIA. Expression of steroidogenic acute regulatory protein (StAR) was monitored by real-time PCR and Western blotting. The results revealed that the increases of progesterone production in the presence of hCG, CT, forskolin and 8-Br-cAMP were augmented by MBP. In contrast, the levels of intracellular cAMP exhibited no statistical significance when MLTC-1 cells were treated as above. These results implied that the site in the steroid biosynthesis pathway affected by MBP occurs after PKA activation in MLTC-1 cells. Moreover, supplementing the medium with 22R-HC and pregnenolone as progesterone precursors for P450 side chain cleavage enzyme (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD), respectively, resulted in no rise in progesterone production, making clear that MBP did not influence the P450scc and 3beta-HSD but on the rate-limiting step, cholesterol transportation into mitochondria. In fact, the above results were confirmed by the upgraded StAR expression in MBP-treated cells. These data support that MBP promotes steroid hormone production by facilitating StAR expression in MLTC-1 cells.     

Enhancement of erythromycin A production with feeding available nitrogen sources in erythromycin biosynthesis phase

Effects of feeding different available nitrogen sources from 80 h in erythromycin biosynthesis phase on the erythromycin A (Er-A) production were investigated in 50 l fermenter. Feeding corn steep liquor and yeast extract, the Er-A production was enhanced, while the biotransformation from erythromycin C (Er-C) to Er-A had no increase. When ammonium sulphate was fed at high feeding rate, the maximal Er-A production and ratio of Er-A to Er-C were 7953 U/ml and 98.18:1 at 184 h, respectively, which were higher than that of the control (6742 U/ml and 5.47:1). The feeding ammonium sulphate process was successfully scaled up from 50 l to 25 m³ fermenter. The maximal Er-A production reached 7938 U/ml at 203 h, which was enhanced by 22.1% compared with the control (6501 U/ml at 192 h). The ratio of Er-A to Er-C was 24.05:1, which was higher than that of the control (4.77:1).     

Pathogenicity Induced by Feline Leukemia Virus, Rickard Strain, Subgroup A Plasmid DNA (pFRA)

A new provirus clone of feline leukemia virus (FeLV), which we named FeLV-A (Rickard) or FRA, was characterized with respect to viral interference group, host range, complete genome sequence, and in vivo pathogenicity in specific-pathogen-free newborn cats. The in vitro studies indicated the virus to be an ecotropic subgroup A FeLV with 98% nucleotide sequence homology to another FeLV-A clone (F6A/61E), which had also been fully sequenced previously. Since subgroup B polytropic FeLVs (FeLV-B) are known to arise via recombination between ecotropic FeLV-A and endogenous FeLV (enFeLV) env elements, the in vivo studies were conducted by direct intradermal inoculation of the FRA plasmid DNA so as to eliminate the possibility of coinoculation of any FeLV-B which may be present in the inoculum prepared by propagating FeLV-A in feline cell cultures. The following observations were made from the in vivo experiments: (i) subgroup conversion from FeLV-A to FeLV-A and FeLV-B, as determined by the interference assay, appeared to occur in plasma between 10 and 16 weeks postinoculation (p.i.); (ii) FeLV-B-like recombinants (rFeLVs), however, could be detected in DNA isolated from buffy coats and bone marrow by PCR as early as 1 to 2 weeks p.i.; (iii) while a mixture of rFeLV species containing various amounts of N-terminal substitution of the endogenous FeLV-derived env sequences were detected at 8 weeks p.i., rFeLV species harboring relatively greater amounts of such substitution appeared to predominate at later infection time points; (iv) the deduced amino acid sequence of rFeLV clones manifested striking similarity to natural FeLV-B isolates, within the mid-SU region of the env sequenced in this work; and (v) four of the five cats, which were kept for determination of tumor incidence, developed thymic lymphosarcomas within 28 to 55 weeks p.i., with all tumor DNAs harboring both FeLV-A and rFeLV proviruses. These results provide direct evidence for how FeLV-B species evolve in vivo from FeLV-A and present a new experimental approach for efficient induction of thymic tumors in cats, which should be useful for the study of retroviral lymphomagenesis in this outbred species.     

BioSig3D: High Content Screening of Three-Dimensional Cell Culture Models

BioSig3D is a computational platform for high-content screening of three-dimensional (3D) cell culture models that are imaged in full 3D volume. It provides an end-to-end solution for designing high content screening assays, based on colony organization that is derived from segmentation of nuclei in each colony. BioSig3D also enables visualization of raw and processed 3D volumetric data for quality control, and integrates advanced bioinformatics analysis. The system consists of multiple computational and annotation modules that are coupled together with a strong use of controlled vocabularies to reduce ambiguities between different users. It is a web-based system that allows users to: design an experiment by defining experimental variables, upload a large set of volumetric images into the system, analyze and visualize the dataset, and either display computed indices as a heatmap, or phenotypic subtypes for heterogeneity analysis, or download computed indices for statistical analysis or integrative biology. BioSig3D has been used to profile baseline colony formations with two experiments: (i) morphogenesis of a panel of human mammary epithelial cell lines (HMEC), and (ii) heterogeneity in colony formation using an immortalized non-transformed cell line. These experiments reveal intrinsic growth properties of well-characterized cell lines that are routinely used for biological studies. BioSig3D is being released with seed datasets and video-based documentation.     

miR-29a suppresses growth and invasion of gastric cancer cells in vitro by targeting VEGF-A

Increasing data shows miR-29a is a key regulator of oncogenic processes. It is significantly down-regulated in some kind of human tumors and possibly functionally linked to cellular proliferation, survival and migration. However, the mechanism remains unclear. In this study, we report miR-29a is significantly under-expressed in gastric cancer compared to the healthy donor. The microvessel density is negatively related to miR-29a expression in gastric cancer tissues. The ectopic expression of miR-29a significantly inhibits proliferation and invasion of gastric cancer cells. Furthermore, western blot combined with the luciferase reporter assays demonstrate that vascular endothelial growth factor A (VEGF-A) is direct target of miR-29a. This is the first time miR-29a was found to suppress the tumor microvessel density in gastric cancer by targeting VEGF-A. Taken together, these results suggest that miR-29a is a tumor suppressor in gastric cancer. Restoration of miR-29a in gastric cancer may be a promising therapeutic approach. [BMB Reports 2014; 47(1):39-44]     

不同林龄油茶人工林土壤酶化学计量及其影响因素

土壤酶化学计量比是揭示微生物生长代谢过程及评价土壤养分资源限制状况的重要指标。油茶是中国南方主要的木本油料作物,近年来愈来愈受关注,但鲜有从生态化学计量学的角度深入理解人工经济林的土壤微生物养分限制状况。本文以亚热带地区不同林龄油茶人工林土壤为研究对象,采用时空互代法在区域尺度上随机选取32个不同林龄油茶人工林并将其分为四个林龄组(9年幼龄林;9—20年近熟林;21—60年成熟林; 60年过熟林),通过测定土壤碳(C)、氮(N)、磷(P)转化酶活性(β-葡糖苷酶(BG)、α-纤维素酶(CBH)、β-乙酰葡糖胺糖苷酶(NAG)、亮氨酸氨基肽酶(LAP)、酸性磷酸酶(AP))及土壤理化因子,探讨不同林龄油茶人工林土壤C、N、P转化酶化学计量特征及其与土壤理化因子的关系。结果表明:五种C、N、P转化酶活性均有随林龄增大而增加的趋势,且AP活性显著高于其它四种酶活性。相关分析结果表明,五种土壤C、N、P转化酶活性均与土壤有机碳和总氮显著相关,与土壤总磷和速效磷含量不相关。土壤酶化学计量比ln(CBH+BG)∶ln(NAG+LAP)、ln(CBH+BG)∶ln(AP)和ln(NAG+LAP)∶ln(AP)均随林龄增大而一定程度增加。亚热带区油茶人工林土壤酶C∶N∶P化学计量比为1∶1∶1.5,这与全球生态系统土壤酶C∶N∶P化学计量比1∶1∶1相偏离,表明亚热带地区油茶人工林土壤微生物生长受磷素限制。冗余分析(RDA)进一步揭示土壤有机碳含量是影响土壤酶活性和酶化学计量比的主要因子。因此,在油茶人工林经营管理中应考虑磷和外源碳的投入,提高土壤微生物酶活性,缓解油茶人工林生态系统的磷限制。研究结果可为亚热带区油茶人工林土壤养分管理和可持续利用提供基础理论支撑。     

Synthesis and biological evaluation of 9-fluorenone derivatives for SPECT imaging of α7-nicotinic acetylcholine receptor

The α7-nicotinic acetylcholine receptor (α7-nAChR) subtype, is found to have a connection with the pathogenesis of a variety of psychiatric and neurological disorders. Herein, we report the development of radioiodinated 9-fluorenone derivatives as single-photon emission computed tomography (SPECT) imaging tracers for α7-nAChRs. Among the derivatives, the best member of the series 10 (Ki = 2.23 nM) were radiolabeled with ¹²⁵I for in vitro and in vivo studies. The radiotracer [¹²⁵I]10 exhibited robust brain uptake and specifically labeled α7-nAChRs with a peak uptake value of 9.49 ± 0.87%ID/g in brain. Blocking studies demonstrated that the tracer was highly specific toward α7-nAChR. Furthermore, ex vivo autoradiography and micro-SPECT/CT dynamic imaging in mice confirmed the excellent imaging properties. In addition, molecular docking was also performed to rationalize the potency of the chosen compounds towards α7-nAChRs. To conclude, compound 10 could serve as a promising radiotracer for the α7-nAChRs.     

Suppressive effect of epigallocatechin‐3‐O‐gallate on endoglin molecular regulation in myocardial fibrosis in vitro and in vivo

Epigallocatechin‐3‐O‐gallate (EGCG), derived from green tea, has been studied extensively because of its diverse physiological and pharmacological properties. This study evaluates the protective effect of EGCG on angiotensin II (Ang II)‐induced endoglin expression in vitro and in vivo. Cardiac fibroblasts (CFs) from the thoracic aorta of adult Wistar rats were cultured and induced with Ang II. Western blotting, Northern blotting, real‐time PCR and promoter activity assay were performed. Ang II increased endoglin expression significantly as compared with control cells. The specific extracellular signal‐regulated kinase inhibitor SP600125 (JNK inhibitor), EGCG (100 μM) and c‐Jun N‐terminal kinase (JNK) siRNA attenuated endoglin proteins following Ang II induction. In addition, pre‐treated Ang II‐induced endoglin with EGCG diminished the binding activity of AP‐1 by electrophoretic mobility shift assay. Moreover, the luciferase assay results revealed that EGCG suppressed the endoglin promoter activity in Ang II‐induced CFs by AP‐1 binding. Finally, EGCG and the JNK inhibitor (SP600125) were found to have attenuated endoglin expression significantly in Ang II‐induced CFs, as determined through confocal microscopy. Following in vivo acute myocardial infarction (AMI)‐related myocardial fibrosis study, as well as immunohistochemical and confocal analyses, after treatment with endoglin siRNA and EGCG (50 mg/kg), the area of myocardial fibrosis reduced by 53.4% and 64.5% and attenuated the left ventricular end‐diastolic and systolic dimensions, and friction shortening in hemodynamic monitor. In conclusion, epigallocatechin‐3‐O‐gallate (EGCG) attenuated the endoglin expression and myocardial fibrosis by anti‐inflammatory effect in vitro and in vivo, the novel suppressive effect was mediated through JNK/AP‐1 pathway.