Updating mRNA variants of the human RSK4 gene and their expression in different stressed situations.

We determined RNA spectrum of the human RSK4 (hRSK4) gene (also called RPS6KA6) and identified 29 novel mRNA variants derived from alternative splicing, which, plus the NCBI-documented ones and the five we reported previously, totaled 50 hRSK4 RNAs that, by our bioinformatics analyses, encode 35 hRSK4 protein isoforms of 35-762 amino acids. Many of the mRNAs are bicistronic or tricistronic for hRSK4. The NCBI-normalized NM_014496.5 and the protein it encodes are designated herein as the Wt-1 mRNA and protein, respectively, whereas the NM_001330512.1 and the long protein it encodes are designated as the Wt-2 mRNA and protein, respectively. Many of the mRNA variants responded differently to different situations of stress, including serum starvation, a febrile temperature, treatment with ethanol or ethanol-extracted clove buds (an herbal medicine), whereas the same stressed situation often caused quite different alterations among different mRNA variants in different cell lines. Mosifloxacin, an antibiotics and also a functional inhibitor of hRSK4, could inhibit the expression of certain hRSK4 mRNA variants. The hRSK4 gene likely uses alternative splicing as a handy tool to adapt to different stressed situations, and the mRNA and protein multiplicities may partly explain the incongruous literature on its expression and comports.

Establishment of New Genetic Markers and Methods for Sex Determination of Mouse and Human Cells using Polymerase Chain Reactions and Crude DNA Samples.

Background: The currently available methods for sexing human or mouse cells have weaknesses. Therefore, it is necessary to establish new methods. Methods: We used bioinformatics approach to identify genes that have alleles on both the X and Y chromosomes of mouse and human genomes and have a region showing a significant difference between the X and Y alleles. We then used polymerase chain reactions (PCR) followed by visualization of the PCR amplicons in agarose gels to establish these genomic regions as genetic sex markers. Results: Our bioinformatics analyses identified eight mouse sex markers and 56 human sex markers that are new, i.e. are previously unreported. Six of the eight mouse markers and 14 of the 56 human markers were verified using PCR and ensuing visualization of the PCR amplicons in agarose gels. Most of the tested and untested sex markers possess significant differences in the molecular weight between the X- and Y-derived PCR amplicons and are thus much better than most, if not all, previously-reported genetic sex markers. We also established several simple and essentially cost-free methods for extraction of crude genomic DNA from cultured cells, blood samples, and tissues that could be used as template for PCR amplification. Conclusion: We have established new sex genetic markers and methods for extracting genomic DNA and for sexing human and mouse cells. Our work may also lend some methodological strategies to the identification of new genetic sex markers for other organismal species.

Modified Si-Miao-San ameliorates pancreatic B cell dysfunction by inhibition of reactive oxygen species-associated inflammation through AMP-kinase activation.

AIM: To observe the effect of modified Si-Miao-San (mSMS) on advanced glycation end products (AGEs)-induced pancreatic B cell dysfunction, as well as examining the underlying mechanisms. METHOD: Pancreatic B cells (INS-1) were stimulated with advanced glycation end products (AGEs, 200 µg·mL(-1)) for 24 h to produce dysfunction in pancreatic B cells and the effects of mSMS observed on insulin secretion, NF-κB (p65) phosphorylation, reactive oxygen species (ROS) production, mitochondria membrane potential (Δψm), cell apoptosis, phosphorylation of AMP-kinase (AMPK), and caspase 3 activity. RESULTS: The AGEs challenge resulted in increased basal insulin secretion, but decreased insulin secretion in response to high glucose, whereas this situation was reversed by mSMS treatment. AGEs stimulation induced NF-κB (p65) phosphorylation and reactive oxygen species (ROS) production, as well as Δψm collapse and cell apoptosis. mSMS inhibited ROS production and inhibited NF-κB activation by attenuating p65 phosphorylation. Meanwhile, AGEs-induced Δψm collapse and cell apoptosis were also reversed by mSMS treatment. Compound C, an inhibitor of AMP-Kinase (AMPK), abolished the beneficial effects of mSMS on the regulation of B cell function, indicating the involvement of AMPK. CONCLUSION: mSMS ameliorated AGEs-induced B cell dysfunction by suppressing ROS-associated inflammation, and this action was related to its beneficial regulation of AMPK activity.

Modified Si-Miao-San inhibits inflammation and promotes glucose disposal in adipocytes through regulation of AMP-kinase.

Modified Si-Miao-San (mSMS) is composed of Rhizoma Coptidis, Cortex Phellodendri, Rhizoma Coptidis Semen Coicis and Atractylodes Rhizome. The prescription is used for the management of diabetes and insulin resistance in the clinic. This study aims to investigate its regulation of glucose disposal in adipocytes. Differentiated 3T3-L1 adipocytes were stimulated with conditioned medium derived from activated macrophages to induce insulin resistance and observed the effects of Mac-CM on insulin-mediated glucose uptake along the insulin receptor substrate-1/PI3K/Akt signaling pathway. Moreover, its regulation of AMPK phosphorylation was also investigated. mSMS enhanced AMPK phosphorylation and promoted basal glucose uptake in adipocytes; mSMS inhibited NF-κB activation by reducing P65 phosphorylation and improved insulin-stimulated IRS-1 tyrosine and Akt phosphorylation, leading to the restoration of insulin-mediated glucose uptake when cells were exposed to inflammatory stimulation. These beneficial effects were diminished in the presence of the AMPK inhibitor compound C. mSMS positively regulated AMPK activity, and this action contributed to improving insulin PI3K signaling by the beneficial regulation of IRS-1 function through inhibition of inflammation in adipocytes.