Preparation and Characterization of Salmon Calcitonin–biotin Conjugates

This study was performed to prepare and characterize the biotinylated Salmon calcitonin (sCT) for oral delivery and evaluate the hypocalcemic effect of biotinylated-sCTs in rats. Biotinylated sCTs was characterized by using high performance liquid chromatography (HPLC) and MALDITOF-MS. The effect of biotinylation on permeability across Caco-2 cell monolayers was examined. Their hypocalcemic effect was determined in rats. Mono- and di-bio-sCTs were separated by reverse phase HPLC. The molecular weights of mono-bio-sCT and di-bio-sCT were determined to be 3,660.5 and 3,900.2 Da, respectively. The permeability of biotinylated-sCTs across Caco-2 cell monolayers was observed with a significant enhancement compared with sCT. Intrajejunal (ij) administration of mono-bio-sCT and di-bio-sCT resulted in sustained reduction in serum calcium levels, with a maximum reduction (% max(d)) of 21.6% and 30% after 4 h and 6 h of application, respectively. The biotin conjugation of sCT may be a promising strategy for increasing the oral bioavailability of sCT and achieving sustained calcium-lowering effects.     

Field experiments on mitigation of harmful algal blooms using a Sophorolipid—Yellow clay mixture and effects on marine plankton

This study examined a new method of mitigating harmful algal blooms (HABs) by combining biosurfactant sophorolipid and yellow clay. To investigate the effects and practicability of this HAB mitigation method, field experiments were carried out during a Cochlodinium bloom near Miruk Island, South Korea, in August 2002. Field experiments examined the effects of sophorolipid and yellow clay on Cochlodinium bloom mitigation and on marine plankton such as bacteriaplankton, heterotrophic protists, and zooplankton. A mixture of 5 mg l⁻¹ sophorolipid and 1 g l⁻¹ yellow clay was sprayed directly on the sea surface and its effect was compared with that of 10 g l⁻¹ of yellow clay applied under similar conditions. The sophorolipid–yellow clay mixture more efficiently mitigated the Cochlodinium bloom (95% removal efficiency after 30 min) than yellow clay alone (79% after 30 min). Further, no variation in bacterial abundance occurred 30 min after spraying the sophorolipid–yellow clay mixture. After 30 min, heterotrophic protist abundance at the surface decreased 21 and 41%, respectively, following the sophorolipid–yellow clay mixture and yellow clay treatments. Zooplankton decreased by 38% 15 min after spraying the mixture and 67% 30 min after spraying the yellow clay. These results indicate that the mixture of sophorolipid and yellow clay had a less adverse effect on bacteriaplankton, heterotrophic protists, and zooplankton than the yellow clay, suggesting that the sophorolipid–yellow clay mixture can mitigate HABs efficiently with fewer negative effects on the pelagic ecosystem.     

The protein interaction network of extracellular vesicles derived from human colorectal cancer cells

Various mammalian cells including tumor cells secrete extracellular vesicles (EVs), otherwise known as exosomes and microvesicles. EVs are nanosized bilayered proteolipids and play multiple roles in intercellular communication. Although many vesicular proteins have been identified, their functional interrelationships and the mechanisms of EV biogenesis remain unknown. By interrogating proteomic data using systems approaches, we have created a protein interaction network of human colorectal cancer cell-derived EVs which comprises 1491 interactions between 957 vesicular proteins. We discovered that EVs have well-connected clusters with several hub proteins similar to other subcellular networks. We also experimentally validated that direct protein interactions between cellular proteins may be involved in protein sorting during EV formation. Moreover, physically and functionally interconnected protein complexes form functional modules involved in EV biogenesis and functions. Specifically, we discovered that SRC signaling plays a major role in EV biogenesis, and confirmed that inhibition of SRC kinase decreased the intracellular biogenesis and cell surface release of EVs. Our study provides global insights into the cargo-sorting, biogenesis, and pathophysiological roles of these complex extracellular organelles.     

Identification, Expression, and Nuclear Location of Murine Mage-b2 Protein, a Tumor-associated Antigen

MAGE-1, which was originally identified by reacting with cytolytic T lymphocytes derived from the blood of melanoma patients, is a member of a gene family consisting of 17 structurally related genes. The MAGE genes are expressed only in the testis among normal tissues and in a number of human tumors of various histological types. Murine MAGE (also called SMAGE or Mage) genes were found in a study aimed at detecting mouse genes homologous to human MAGE genes. However, the biological functions of MAGE and Mage are currently unknown. To understand the biological functions of Mage, in the present study a recombinant SMAGE2 (Mage-b2) protein of 43 kDa was produced and monoclonal antibodies reactive with Mage-b2 protein were generated. One monoclonal antibody, smpG4A, specifically recognized a 43 kDa protein in lysates of Mage-b2 mRNA-positive sarcoma cells and of the testis. Immunohistochemistry showed that Mage-b2 is located in the nucleus of Mage-b2 mRNA-positive sarcoma cells. These results should contribute to understanding the biological functions of Mage.     

Single nucleotide polymorphisms and the linkage disequilibrium at the LDL receptor gene in Koreans

The low-density lipoprotein (LDL) receptor gene is one of the important genes in cardiovascular biology, but gives difficulty in single nucleotide polymorphism (SNP) marker choice. Twelve SNPs at the LDL receptor locus were genotyped in 117 Korean individuals. Six SNPs (rs8111982, rs12983082, rs1003723, rs5925, rs6413504, and rs2738464) were polymorphic in the Korean sample. And 8 SNPs (rs5931, rs811 1982, rs12983082, rs11669576, rs1003723, rs5925, rs641 3504, and rs2738464) were polymorphic in at least one population among the five ethnic groups (European: CEU; Chinese: CHB; Japanese: JPT; African:YRI; and Korean: KOR). Phylogenetic trees constructed based on the twelve SNPs showed a pattern clustering KOR with CHB and JPT as more closely related than either YRI or CEU. The plot of the pairwise linkage disequilibrium (LD) values for the exon 13 SNP (rs5925) against the physical distances between pairs of SNPs showed a tendency of LD decrease in the upstream of rs5925, while the low r² values are present in the downstream of rs5925 (≥0.20). The three SNPs (rs12983082, rs1003723, and rs5925) which are ethnically polymorphic are likely to be useful SNP markers for the LDL receptor locus. The interblock region between exon 13 and exon 18 delimited by the less stringent block definition (four gamete rule) contains a feasible recombination hotspot in the LDL receptor gene in Koreans.     

Regulation of magnesium-inhibited cation current by actin cytoskeleton rearrangement

Magnesium-inhibited, non-selective cation current (I(MIC)) is activated by depletion of intracellular Mg(2+) and ATP. I(MIC) transports various divalent cations including Mg(2+) and Ca(2+), and is involved in cell viability. We investigated the effect of actin dynamics on I(MIC). Formation of a stable cortical actin network by calyculin A inhibited the activation of I(MIC), while the actin depolymerizing reagent, cytochalasin D, reversed the inhibition. Induction of a dense cortical actin layer by transfecting the constitutively active form of RhoA also inhibited the activation of I(MIC). These results suggest that the activation of I(MIC) may be dynamically regulated by actin cytoskeleton rearrangement.     

Functional analysis of six drought-inducible promoters in transgenic rice plants throughout all stages of plant growth

There are few efficient promoters for use with stress-inducible gene expression in plants, and in particular for monocotyledonous crops. Here, we report the identification of six genes, Rab21, Wsi18, Lea3, Uge1, Dip1, and R1G1B that were induced by drought stress in rice microarray experiments. Gene promoters were linked to the gfp reporter and their activities were analyzed in transgenic rice plants throughout all stages of plant growth, from dry seeds to vegetative tissues to flowers, both before and after drought treatments. In fold induction levels, Rab21 and Wsi18 promoters ranged from 65- and 36-fold in leaves to 1,355- and 492-fold in flowers, respectively, whereas Lea3 and Uge1 were higher in leaves, but lower in roots and flowers, as compared with Rab21 and Wsi18. Dip1 and R1G1B promoters had higher basal levels of activity under normal growth conditions in all tissues, resulting in smaller fold-induction levels than those of the others. In drought treatment time course, activities of Dip1 and R1G1B promoters rapidly increased, peaked at 2 h, and remained constant until 8 h, while that of Lea3 slowly yet steadily increased until 8 h. Interestingly, Rab21 activity increased rapidly and steadily in response to drought stress until expression peaked at 8 h. Thus, we have isolated and characterized six rice promoters that are all distinct in fold induction, tissue specificity, and induction kinetics under drought conditions, providing a variety of drought-inducible promoters for crop biotechnology.