Ovarian substance induces steroid production in cultured granulosa cells

Summary The rat ovary produces an apparently low molecular weight substance that mimics the action of follitropin (FSH) on ovarian granulosa cells in culture. Similar to FSH action, the ovarian substance (OS) induces temporal cell rounding and, later on, intensive progestin production. However, unlike FSH, OS does not induce accumulation of cyclic AMP (cAMP) in the granulosa cells. The ovarian factor cannot be cAMP as its action is not abolished by phosphodiesterase (PDE) treatment. Neither is it a possible PDE inhibitor, as it does not augment cAMP accumulation in granulosa cells or Friend erythroleukemic cells induced by FSH or PGE₁, respectively. The factor is still active after heating for 20 min at 90° C but is rapidly inactivated by alkali treatment. In addition, treatment with various proteases did not abolish the steroidogenic activity. These findings suggest a possible novel intraovarian regulator of the granulosa cell function. Presented in the symposium on Plant and Animal Physiology in Vitro at the 33rd Annual Meeting of the Tissue Culture Association, San Diego, California, June 6–10, 1982. This work was supported by the United States-Israel Binational Science Foundation, Grant 2656/81. This symposium was supported in part by the following organizations: Bellco Glass, Inc., California Branch of the Tissue Culture Association, Collaborative Research, Hana Media, Hybridtech, K C Biological, Inc., and Millipore Corporation.     

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

LncRNA LEF1-AS1 regulates the migration and proliferation of vascular smooth muscle cells by targeting miR-544a/PTEN axis

Long noncoding RNAs (lncRNAs) play important roles in endothelium development. A lncRNA, LEF1-AS1, is recently emerging as a potent mediator of the proliferation and migration of a number of cells, including smooth muscle cells. However, the effects of LEF1-AS1 in atherosclerosis remains largely unknown. Specimens from patients with coronary artery atherosclerosis were collected. The quantitative real-time polymerase chain reaction was used to analyze levels of LEF1-AS1 and microRNA-544a (miR-544a). Western blot analysis was used to assess PTEN, P-Akt, and T-Akt protein expression. Proliferation, migration, and invasion of cells were analyzed by cell counting kit-8 assay, scratch wound assay, and transwell assay, respectively. The interaction between LEF1-AS1, miR-544a, and PTEN was probed using bioinformatical analysis and dual-luciferase assay. In plasma and tissue of patients with coronary artery atherosclerosis, LEF1-AS1 was upregulated and miR-544a was downregulated. A negative correlation was found between LEF1-AS1 and miR-544a. miR-544a overexpression reversed the inhibition of LEF1-AS1 in smooth muscle cell proliferation and invasion, which were mediated through the PTEN pathway. LEF1-AS1 regulates smooth muscle cell proliferation and migration through the miR-544a/PTEN axis, indicating that LEF1-AS1 may be a potential therapeutic target in atherosclerosis.     

Anti-inflammatory effect of l-cysteine (a semi-essential amino acid) on 5-FU-induced oral mucositis in hamsters

Oral mucositis is an inflammation of the oral mucosa mainly resulting from the cytotoxic effect of 5-fluorouracil (5-FU). The literature shows anti-inflammatory action of l-cysteine (l-cys) involving hydrogen sulfide (H₂S). In view of these properties, we investigate the effect of l-cys in oral mucositis induced by 5-FU in hamsters. The animals were divided into the following groups: saline 0.9%, mechanical trauma, 5-FU 60–40 mg/kg, l-cys 10/40 mg and NaHS 27 µg/kg. 5-FU was administered on days 1st to 2nd; 4th day excoriations were made on the mucosa; 5th–6th received l-cys and NaHS. For data analysis, histological analyses, mast cell count, inflammatory and antioxidants markers, and immunohistochemistry (cyclooxygenase-2(COX-2)/inducible nitric oxide synthase (iNOs)/H₂S) were performed. Results showed that l-cys decreased levels of inflammatory markers, mast cells, levels of COX-2, iNOS and increased levels of antioxidants markers and H₂S when compared to the group 5-FU (p < 0.005). It is suggested that l-cys increases the H₂S production with anti-inflammatory action in the 5-FU lesion.

     

Low Density Lipoproteins Promote Unstable Calcium Handling Accompanied by Reduced SERCA2 and Connexin-40 Expression in Cardiomyocytes

The damaging effects of high plasma levels of cholesterol in the cardiovascular system are widely known, but little attention has been paid to direct effects on cardiomyocyte function. We therefore aimed at testing the hypothesis that Low Density Lipoprotein (LDL) cholesterol affects calcium dynamics and signal propagation in cultured atrial myocytes. For this purpose, mRNA and protein expression levels were determined by real time PCR and western blot analysis, respectively, and intracellular calcium was visualized in fluo-4 loaded atrial HL-1 myocyte cultures subjected to field stimulation. At low stimulation frequencies all cultures had uniform calcium transients at all tested LDL concentrations. However, 500 µg LDL/mL maximally reduced the calcium transient amplitude by 43% from 0.30±0.04 to 0.17±0.02 (p<0.05). Moreover, LDL-cholesterol dose-dependently increased the fraction of alternating and irregular beat-to-beat responses observed when the stimulation interval was shortened. This effect was linked to a concurrent reduction in SERCA2, RyR2, IP3RI and IP3RII mRNA levels. SERCA2 protein levels were also reduced by 43% at 200 µg LDL/mL (p<0.05) and SR calcium loading was reduced by 38±6% (p<0.001). By contrast, HDL-cholesterol had no significant effect on SERCA expression or SR calcium loading. LDL-cholesterol also slowed the conduction velocity of the calcium signal from 3.2+0.2 mm/s without LDL to 1.7±0.1 mm/s with 500 µg LDL/mL (p<0.05). This coincided with a reduction in Cx40 expression (by 44±3%; p<0.05 for mRNA and by 79±2%; p<0.05 for Cx40 protein at 200 µg/ml LDL) whereas the Cx-43 expression did not significantly change. In conclusion, LDL-cholesterol destabilizes calcium handling in cultured atrial myocytes subjected to rapid pacing by reducing SERCA2 and Cx40 expression and by slowing the conduction velocity of the calcium signal.     

Molecular Characterization of E-Type Prostanoid Receptor 4 (EP4) from Ayu (Plecoglossus altivelis) and Its Functional Analysis in the Monocytes/Macrophages

Prostaglandin E2 (PGE2) plays an important role in a broad spectrum of physiological and pathological processes by interacting with E-type prostanoid receptors (EPs). EP4 is one of four EP subtypes known to mediate the immune response in mammalian monocytes/macrophages. However, the precise function and characteristics of EP4 in fish remain unclear. In this study, we characterized a novel EP4-like (PaEP4L) gene from ayu, Plecoglossus altivelis. The cDNA sequence of PaEP4L is 2781 nucleotides (nts) in length, encoding a polypeptide of 459 amino acid residues with a calculated molecular weight of 51.17 kDa. Sequence comparison and phylogenetic tree analysis showed that PaEP4L shared 76% amino acid identity with that of the Atlantic salmon (Salmo salar). PaEP4L mRNA was detected by real-time quantitative PCR (QPCR) in all tested tissues and head kidney-derived monocytes/macrophages (MO/MФ). It varied greatly in liver, spleen and MO/MФ upon Vibrio anguillarum infection. Western blot analysis revealed a significant increase of PaEP4L in cell homogenates from ayu MO/MФ upon V. anguillarum infection. Moreover, anti-PaEP4L IgG reversed the down-regulation of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) mRNA expression as well as phagocytosis in ayu MO/MФ caused by PGE2. There were no significant differences in the respiratory burst response between PGE2 treated and untreated cells. We further found that cAMP mediated PGE2/PaEP4L signal in ayu MO/MФ. In conclusion, our results indicate that PaEP4L mediates PGE2 effects on ayu MO/MФ function, revealing that EP4 also plays a role in the modulation of cells of the fish’s innate immune system.     

Kinase–substrate Edge Biomarkers Provide A More Accurate Prognostic Prediction in ER-negative Breast Cancer

The estrogen receptor (ER)-negative breast cancer subtype is aggressive with few treatment options available. To identify specific prognostic factors for ER-negative breast cancer, this study included 705,729 and 1034 breast invasive cancer patients from the Surveillance, Epidemiology, and End Results (SEER) and The Cancer Genome Atlas (TCGA) databases, respectively. To identify key differential kinase–substrate node and edge biomarkers between ER-negative and ER-positive breast cancer patients, we adopted a network-based method using correlation coefficients between molecular pairs in the kinase regulatory network. Integrated analysis of the clinical and molecular data revealed the significant prognostic power of kinase–substrate node and edge features for both subtypes of breast cancer. Two promising kinase–substrate edge features, CSNK1A1–NFATC3 and SRC–OCLN, were identified for more accurate prognostic prediction in ER-negative breast cancer patients.     

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.