Subcellular localization and expression analysis of the BmDSCLP protein from silkworm, Bombyx mori

Leucine-rich repeat (LRR) proteins play important roles in the transduction of cellular signals and activation of defense responses. By scanning the cDNA library of silkworm (Bombyx mori) pupae constructed in our laboratory, we identified a 1557 bp gene that encodes a protein homologous to the death-associated small cytoplasmic leucine-rich protein, which was named as BmDSCLP. The full-length gene (GenBank accession no. FJ602779) contained a 642 bp open reading frame (ORF) encoding 213 amino acid residues. The ORF of this gene was inserted into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression plasmid and the fusion protein was expressed in Escherichia coli BL21(DE3) cells. The fusion protein was purified by Ni-affinity chromatography and fast protein liquid chromatography (FPLC) and its size was then, determined by liquid chromatography-mass spectrometry (LC/MS/MS) and found to be 27.74 kD. Polyclonal antibodies were raised by subcutaneous injection of the recombinant protein into New Zealand white rabbits and the titer reached 1:12800. Analysis of the subcellular localization of the BmDSCLP protein revealed that, the protein was localized in both the cytoplasm and nucleus, but the amount in the former was slightly higher than that in the latter. In addition, real-time fluorescence quantification polymerase chain reaction studies were conducted to investigate BmDSCLP transcription at different developmental stages and in different tissues of the fifth instar larva. The results indicated that, BmDSCLP is widely transcribed in different stages and tissues of the silkworm. Analysis of stage-specific transcription patterns indicated that, the transcriptional level of BmDSCLP was highest in adults and lowest in eggs. Analysis of tissue-specific transcription patterns revealed that, the transcriptional level of BmDSCLP was highest in genital organs and lowest in silk glands. These results suggest that BmDSCLP plays important roles in the reproductive development of B. mori.Keywords: Bombyx mori, death-associated small cytoplasmic leucine-rich protein, prokaryotic expression, fluorescence quantification polymerase chain reactio

MiR-214 Attenuates Osteogenic Differentiation of Mesenchymal Stem Cells via Targeting FGFR1

Background/Aims: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. Methods: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. Results: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. Conclusions: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis

Schwann Cells Transplantation Improves Locomotor Recovery in Rat Models with Spinal Cord Injury: a Systematic Review and Meta-Analysis

Background/Aims: Schwann cells (SCs) which were demonstrated to be responsible for axonal myelination and ensheathing are widely studied and commonly used for cell transplantation to treat spinal cord injury (SCI). We performed this meta-analysis to summarize the effects of SCs versus controls for locomotor recovery in rat models of traumatic SCI. Methods: Studies of the BBB scores after transplantation of SCs were searched out from Pubmed, Cochrane Library Medline databases and analyzed by Review Manager 5.2.5. Results: Thirteen randomized controlled animal trials were selected with 283 rats enrolled. The studies were divided to different subgroups by different models of SCI, different cell doses for transplantation, different sources of SCs and different transplantation ways. The pooled results of this meta-analysis suggested that SCs transplantation cannot significantly improve the locomotor recovery at a short time after intervention (1 week after transplantation) in both impacted and hemi-sected SCI models. However, at a longer time after intervention (3, 5-7 and over 8 weeks after transplantation), significant improvement of BBB score emerged in SCs groups compared with control groups. Subgroup analyses revealed that SCs transplantation can significantly promote locomotor recovery regardless of in high or low doses of cells, from different sources (isolated from sciatic nerves or differentiated from bone marrow stromal cells(BMSCs)) and with or without scaffolding. Conclusion: SCs seem to demonstrate substantial beneficial effects on locomotor recovery in a widely-used animal models of SCI

Conserved expression of natural antisense transcripts in mammals

BACKGROUND: Recent studies had found thousands of natural antisense transcripts originating from the same genomic loci of protein coding genes but from the opposite strand. It is unclear whether the majority of antisense transcripts are functional or merely transcriptional noise. RESULTS: Using the Affymetrix Exon array with a modified cDNA synthesis protocol that enables genome-wide detection of antisense transcription, we conducted large-scale expression analysis of antisense transcripts in nine corresponding tissues from human, mouse and rat. We detected thousands of antisense transcripts, some of which show tissue-specific expression that could be subjected to further study for their potential function in the corresponding tissues/organs. The expression patterns of many antisense transcripts are conserved across species, suggesting selective pressure on these transcripts. When compared to protein-coding genes, antisense transcripts show a lesser degree of expression conservation. We also found a positive correlation between the sense and antisense expression across tissues. CONCLUSION: Our results suggest that natural antisense transcripts are subjected to selective pressure but to a lesser degree compared to sense transcripts in mammals

Cell Active Hydroxylactam Inhibitors of Human Lactate Dehydrogenase with Oral Bioavailability in Mice

A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 μM lactate IC₅₀ in a MiaPaca2 cell line. Optimization of the series led to <b>29</b>, a potent cell active molecule (MiaPaca2 IC₅₀ = 0.67 μM) that also possessed good exposure when dosed orally to mice