In the present study, we investigated the effect of 70% EtOH extract from Hippophae Rhamnoides L. leaves (HRL) on the anti-obesity effect in 3T3-L1 cells. The effects of HRL on lipid accumulation in 3T3-L1 cells were examined using Oil Red O staining. In addition, we examined the gene expression levels by using RT-PCR and western blot. The results of this analysis showed that 100 ㎍/㎖ HRL significantly increased the inhibition of lipid accumulation by 82.25%; significantly decreased the mRNA expression of sterol regulatory element binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding proteins α (C/EBPα), and fatty acid synthase (FAS) in 3T3-L1 cells as well as the stimulated protein expression of AMP-activated protein kinase (AMPK); and suppressed the expression level of PPARγ. These results suggest that HRL can prevent adipogenesis through activation of AMPKα and inhibition of adipogenesis transcription factors.

Throughout their meiotic maturation in most mammals, oocytes are arrested twice, prophase I and metaphase II. Being released from these arrests, transient or oscillation of intracellular Ca2+ concentration is observed in the ooplasm, which is not answered in relation to the specific role in the resumption of meiotic arrest. Recently, Ca2+/calmodulin-dependent protein kinase II (CaM KII) has been known as a Ca2+ oscillation decoder from the in vitro experiment. CaM KII is multifunctional serine/threonine kinase observed in most cells. Present studies were performed to investigate the role of CaM KII during resumption of meiotic arrest and activation in vitro of mouse oocytes. It was questioned whether CaM KII might be involved in the meiotic resumption of mouse oocytes. Compared to the control, both of CaM KII inhibitors, KN-93 and KN-62, significantly inhibited germinal vesicle breakdown (GVBD) of mouse oocytes in a dose-dependent manner. As the concentration of KN-93 increased, concomitant decrease of intracellular Ca2+ concentration ([Ca2+]i) was also observed using confocal laser scanning microscope (CLSM) and an intracellular Ca2+ indicator, fluo 3-AM. When GVBD oocytes were treated with 6% ethanol, small [Ca2+]i transient was observed in oocytes bathed with Ca2+-free medium and large increase was observed in oocytes bathed with Ca2+-containing medium, suggesting that [Ca2+]i transient could happen from intracellular Ca2+ store as well as Ca2+ influx through Ca2+-channel on the oolemma. However, KN-93 inhibited the [Ca2+]i transient of GVBD oocytes in both cases. Using monoclonal antibodies against α-subunit of CaM KII, tubulin and microtubule-assocaited proteins (MAPs), CaM KII has been colocalized on the spindle with tubulin and MAPs. The present study also demonstrated the presence of α-subunit of CaM KII in heart, kidney, testes, ovary as well as in brain of the mouse. In ovarian follicles, CaM KII was expressed in granulosa cells and oocytes. Based on overall the above results, followings are suggested. First, CaM KII might be involved in the regulatory mechanism of meiotic resumption. Second, CaM KII might play a regulatory role in the stabilization of microtubule.

Since global climate changes drastically, pre-harvest sprouting (PHS) is expected to pose serious problems in rice production. CBL-interacting serine/threonine protein kinases (CIPKs) have been implicated to play important role in regulating various abiotic stresses such as cold, salinity and drought. In this study, to understand the function of this gene under pre-harvest sprouting in rice, a cDNA clone encoding CBL-interacting protein kinase 15 (CIPK15) was isolated from rice flowers. This gene is 2,818 bp long with 1,332 bp coding region that encodes a polypeptide of 443 amino acids. We constructed a recombinant vector carrying the OsCIPK15 under the control of the CaMV 35S promoter and Tnos terminator and transformed into rice using Agrobacterium tumefaciens. Insertion of the gene was verified in transformants using HPT resistance test and genomic PCR. Transcriptional profiling using tissues of wild type, Gopum, revealed expression of the gene in whole plant tissues with level of expression highest in the seeds suggesting possible role in dormancy. Comparative expression analysis of the gene in transgenic and wild type through semi-quantitative RT-PCR and real-time PCR showed higher expression in transgenic rice lines. Moreover, screening in the mist chamber showed overexpression lines that were resistant to the PHS. This result suggests the involvement of OsCIPK15 in the regulation of pre-harvest sprouting.

A cDNA clone encoding CBL-interacting protein kinase 1 (CIPK1) was isolated from Chinese cabbage seedlings. The gene, BrCIPK1 consisted of 1,982 bp long with 216 bp of the 5’-untranslated region (UTR), 1,509 bp of the coding region and 257 bp of the 3’-UTR. It is highly conserved CBL-interacting module with absolutely conserved domain among the 15 amino acid NAF domain of the 15 related genes. Southern blot analysis showed a single copy number. BrCIPK1 gene was localized in the cytoplasm and peripheral region in the plant cell which is highly expressed in seedling of rice and in the shoot and pistil of Arabidopsis. Analyses of gene expression on Ubi-1::BrCIPK1 rice lines was differentially accumulated by cold, salinity and drought, indicating its biological roles in the multiple stress response pathways in plants. Further, the expression of BrCIPK1 is hijacked by rice calcineurin-B-like protein (OsCBL5). Moreover, mRNA expression of P5CS1, a gene responsible for proline biosynthesis is regulated by the BrCIPK1 during abiotic stresses resulting to improved accumulation of proline. The interaction of BrCIPK1 with OsCBL5 along with the regulation of P5CS1 explained the enhanced tolerance of transgenic rice. This gene could be used in the development of rice varieties with enhanced tolerance to abiotic stresses.

A cDNA clone encoding CBL-interacting protein kinase 1 (CIPK1) was isolated from Chinese cabbage seedlings. The gene, BrCIPK1 consisted of 1,982 bp long with 216 bp of the 5’-untranslated region (UTR), 1,509 bp of the coding region and 257 bp of the 3’-UTR. It is highly conserved CBL-interacting module with absolutely conserved domain among the 15 amino acid NAF domain of the 15 related genes. Southern blot analysis showed a single copy number. BrCIPK1 gene was localized in the cytoplasm and peripheral region in the plant cell which is highly expressed in seedling of rice and in the shoot and pistil of Arabidopsis. Analyses of gene expression on Ubi-1::BrCIPK1 rice lines was differentially accumulated by cold, salinity and drought, indicating its biological roles in the multiple stress response pathways in plants. Further, the expression of BrCIPK1 is hijacked by rice calcineurin-B-like protein (OsCBL5). Moreover, mRNA expression of P5CS1, a gene responsible for proline biosynthesis is regulated by the BrCIPK1 during abiotic stresses resulting to improved accumulation of proline. The interaction of BrCIPK1 with OsCBL5 along with the regulation of P5CS1 explained the enhanced tolerance of transgenic rice. This gene could be used in the development of rice varieties with enhanced tolerance to abiotic stresses.

Change in intracellular -concentration () is an essential event for egg activation and further development. ion is originated from intracellular -store via inositol 1,4,5-triphosphate receptor and/or influx via channel. This study was performed to investigate whether changes in /calmodulin dependent protein kinase II (CaM KII) activity affect influx during artificial egg activation with ethanol using monitoring system and whole-cell patch clamp technique. Under ion-omitted condition, -oscillation was stopped within 30 min post microinjection of porcine sperm factor, and ethanol-induced increase was reduced. To investigate the role of CaM KII known as an integrator of - oscillation during mammalian egg fertilization, CaM KII activity was tested with a specific inhibitor KN-93. In the eggs treated with KN-93, ethanol failed to induce egg activation. In addition, KN-93 inhibited inward current () in a time-dependent manner in whole-cell configuration. Immunostaining data showed that the voltage-dependent channels were distributed along the plasma membrane of mouse egg and 2-cell embryo. From these results, we suggest that influx during fertilization might be controlled by CaM KII activity.