Coupling of the inositol 1,4,5-trisphosphate receptor and chromogranins A and B in secretory granules

The secretory granules of neuroendocrine cells which contain large amounts of Ca(2+) and chromogranins have been demonstrated to release Ca(2+) in response to inositol 1,4,5-trisphosphate (IP(3)). Moreover, chromogranin A (CGA) has been shown to interact with several secretory granule membrane proteins, including the IP(3) receptor (IP(3)R). To determine whether the IP(3)Rs interact directly with chromogranins A and B (CGB), two major proteins of the secretory granules, we have used purified IP(3)R from bovine cerebellum in the interaction study with CGA and CGB, and have shown that chromogranins A and B directly interact with the IP(3)R at the intravesicular pH 5.5. Immunogold cytochemical study using the IP(3)R and CGA antibodies indicated that IP(3)R-labeled gold particles were localized in the periphery of the secretory granules, indicating the presence of the IP(3)Rs on the secretory granule membrane. To determine whether the IP(3)R and chromogranins A and B are physically linked in the cells, bovine type 1 IP(3)R (IP(3)R-1) and CGA or CGB are co-transfected into COS-7 cells and co-immunoprecipitation was carried out. Immunoprecipitation of the cell extracts demonstrated the presence of CGA-IP(3)R-1 and CGB-IP(3)R-1 complexes, respectively, indicating the complex formation between the IP(3)R and chromogranins A and B in native state.     

The origin of the domestic pig: independent domestication and subsequent introgression

The domestic pig originates from the Eurasian wild boar (Sus scrofa). We have sequenced mitochondrial DNA and nuclear genes from wild and domestic pigs from Asia and Europe. Clear evidence was obtained for domestication to have occurred independently from wild boar subspecies in Europe and Asia. The time since divergence of the ancestral forms was estimated at approximately 500,000 years, well before domestication approximately 9,000 years ago. Historical records indicate that Asian pigs were introduced into Europe during the 18th and early 19th centuries. We found molecular evidence for this introgression and the data indicated a hybrid origin of some major "European" pig breeds. The study is an advance in pig genetics and has important implications for the maintenance and utilization of genetic diversity in this livestock species.     

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase gene are resistant to diphenyl ether herbicide oxyfluorfen

Protoporphyrinogen oxidase (Protox), the penultimate step enzyme of the branch point for the biosynthetic pathway of Chl and hemes, is the target site of action of diphenyl ether (DPE) herbicides. However, Bacillus subtilis Protox is known to be resistant to the herbicides. In order to develop the herbicide-resistant plants, the transgenic rice plants were generated via expression of B. subtilis Protox gene under ubiquitin promoter targeted to the cytoplasm or to the plastid using Agrobacterium-mediated gene transformation. The integration and expression of the transgene were investigated at T0 generation by DNA and RNA blots. Most transgenic rice plants revealed one copy transgene insertion into the rice genome, but some with 3 copies. The expression levels of B. subtilis Protox mRNA appeared to correlate with the copy number. Furthermore, the plastidal transgenic lines exhibited much higher expression of the Protox mRNA than the cytoplasmic transgenic lines. The transgenic plants expressing the B. subtilis Protox gene at T0 generation were found to be resistant to oxyfluorfen when judged by cellular damage with respect to cellular leakage, Chl loss, and lipid peroxidation. The transgenic rice plants targeted to the plastid exhibited higher resistance to the herbicide than the transgenic plants targeted to the cytoplasm. In addition, possible resistance mechanisms in the transgenic plants to DPE herbicides are discussed.     

Rhodeus pseudosericeus sp. nov., a new bitterling from South Korea (Cyprinidae, Acheilognathinae)

A new bitterling, Rhodeus pseudosericeus sp. nov., is described on the basis of 31 specimens from five localities included in the Namhan River system, South Korea. The new species is distinguished from other Rhodeus species by the following combination of characters: branched dorsal fin rays 9–10 (mode 9); branched anal fin rays 9–11 (mode 10); longest simple ray of dorsal fin strong and stiff, distally segmented; pelvic fin rays i, 6–7; iris of males blackish; dorsal and anal fins of males grayish in breeding season; karyotype with 2n = 48 (8m + 20sm + 20st). Rhodeus pseudosericeus sp. nov. is similar to Rhodeus sericeus sericeus in the number of pelvic fin and branched dorsal fin rays and the melanophores present on the dorsal fin membrane, but differs from the latter in having a greater body depth, more branched anal fin rays, fewer vertebrae, a lower number of scales in the lateral series, and differing male nuptial coloration. Received: June 30, 2000 / Revised: February 21, 2001 / Accepted: March 6, 2001     

Recycling wasted biomaterial,crab shells,as an adsorbent for the removal of high concentration of phosphate

In this study, crab shells were recycled as an adsorbent for the removal of phosphate. The effects of shell particle size, temperature, pH and phosphate concentration on phosphate removal were investigated. Shell particles less than 1000 μm in diameter removed more than 85% of 500 mg/L phosphate in 24 h while particles 3350 μm in diameter exhibited only 50% removal efficiency. Temperature showed negligible effect on phosphate removal in the range of 15–45 °C. Although removal efficiency was highest at pH 2.0, the efficiency remained 50–60% at pH of 4.0–10.0. The maximum removal capacity was calculated as 108.9 mg/g through Langmuir isotherm plotting, which was 17.0 and 4.7 times higher than those of coal fly ash and scallop shells, respectively. Although calcium carbonate played an active role in the removal of phosphate, both proteins composing 12.5% of crab shells and cellulose-like backbone of the crab shells also played an important role in phosphate removal.     

NGF-induced moesin phosphorylation is mediated by the PI3K,Rac1 and Akt and required for neurite formation in PC12 cells

Moesin is a member of ERM family proteins which act as the cross-linkers between plasma membrane and actin-cytoskeleton and is activated by phosphorylation at Thr-558. In neurons, suppression of radixin and moesin alters the growth cone morphology. However, the significance of phosphorylation of ERM proteins in neuronal cells has not been fully investigated. In this study, we studied the signaling pathways responsible for moesin phosphorylation and its functional importance in NGF-treated PC12 cells. NGF rapidly induced the phosphorylation of moesin at Thr-558 in PC12 cells which was dependent on PI3K and Rac1. We found that Akt interacted and phosphorylated with moesin both in vitro and in vivo. Inhibition of PI3K and Rac1 abolished the NGF-induced Akt activation, indicating that Akt is at the downstream of PI3K and Rac1. To examine the functional role of phosphorylated ERM proteins, a dominant negative mutant form of moesin (T558A) was introduced into PC12 cells. The mutant significantly reduced the frequency of cells with neurites following NGF treatment. Our results indicate that PI3K, Rac1 and Akt-dependent phosphorylation of moesin is required for the NGF-induced neurite formation in differentiating PC12 cells.     

Comparison of PERV genomic locations between Asian and European pigs

Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation.     

Activation of RhoA and FAK induces ERK-mediated osteopontin expression in mechanical force-subjected periodontal ligament fibroblasts

The precise mechanism by which Rho kinase translates the mechanical signals into OPN up-regulation in force-exposed fibroblasts has not been elucidated. Human periodontal ligament fibroblasts (hPLFs) were exposed to mechanical force by centrifuging the culture plates at a magnitude of 50 g/cm² for 60 min. At various times of the force application, they were processed for analyzing cell viability, trypan blue exclusion, and OPN expression at protein and RNA levels. Cellular mechanism(s) of the force-induced OPN up-regulation was also examined using various kinase inhibitors or antisense oligonucleotides specific to mechanosensitive factors. Centrifugal force up-regulated OPN expression and induced a rapid and transient increase in the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), and Elk1. Pharmacological blockade of RhoA/Rho-associated coiled coil-containing kinase (ROCK) signaling markedly reduced force-induced FAK and ERK1/2 phosphorylation. Transfecting hPLFs with FAK antisense oligonucleotide diminished ERK1/2 activation and force-induced OPN expression. Further, ERK inhibitor inhibited significantly OPN expression, Elk1 phosphorylation, and activator protein-1 (AP-1)-DNA binding activation, but not FAK phosphorylation, in the force-applied cells. These results demonstrate that FAK signaling plays critical roles in force-induced OPN expression in hPLFs through interaction with Rho/ROCK as upstream effectors and ERK-Elk1/ERK-c-Fos as downstream effectors.     

Distinct roles of vascular endothelial growth factor receptor-1- and receptor-2-mediated signaling in T cell priming and Th17 polarization to lipopolysaccharide-containing allergens in the lung

Vascular endothelial growth factor (VEGF) is a key mediator in the development of airway immune dysfunction to inhaled allergens. However, the exact role of its receptors-mediated signaling is controversial. In this study, we evaluated the role of VEGF receptor (VEGFR)-1- and VEGFR-2-mediated signaling in T cell priming and polarization in the context of inhalation of LPS-containing allergens. A murine asthma model of mixed Th1 and Th17 cell responses was generated using intranasal sensitization with LPS-containing allergens. Pharmacologic intervention was performed during sensitization. In vivo production of VEGF and Th1- and Th17-polarizing cytokines (IL-12p70 and IL-6, respectively) were upregulated by airway exposure to LPS. Pharmacological intervention with a VEGFR-2-neutralizing Ab (anti-Flk1 mAb) abolished the production of IL-6 (but not IL-12p70) and the subsequent development of allergen-specific Th17 cell response. On the other hand, blocking VEGFR-1 signaling with a VEGFR-1 antagonist (anti-Flt1 hexapeptide) did not affect the production of IL-12p70 and IL-6. However, blocking VEGFR-1 signaling resulted in T cell tolerance rather than priming, mainly by inhibiting the maturation of lung dendritic cells, and their migration into lung-draining lymph nodes. These results suggest that T cell priming to LPS-containing allergens depends on VEGFR-1-mediated signaling, and the subsequent Th17 polarization depends on VEGFR-2 signaling.