Changes in actin-related gelation of crude cell extracts during differentiation of myeloid leukemia cells

Gelation of extracts of a myeloid leukemia cell line (Ml) was compared before and after differentiation induced with conditioned medium (CM) from rat embryo cells. Although an extract of Mml cells, a macrophage line derived from Ml line, gelled when warmed in the presence of 2 mM MgCl2, undifferentiated Ml cells gelled only after dialysis and a supplement of exogenous actin. After differentiation had been induced, an addition of exogenous actin, but not dialysis, was needed for gelation. Small amounts of KCl always inhibited the gelation of the control Ml cell extracts, but they promoted gelation of the CM-treated Ml and Mml cell extracts. Thus, the dialysis required for gelation of the control Ml cell extract appears to be necessary for the exclusion of endogenous KCl. Several possible mechanisms for the KCl control of gelation, as well as different requirements of exogenous actin needed for gelation are discussed based on the results of our experiments.     

Epidermal growth factor increases clathrin-dependent endocytosis and degradation of claudin-2 protein in MDCK II cells

Epidermal growth factor (EGF) decreases the mRNA and protein levels of claudin-2 (CLDN2) in Madin-Darby canine kidney (MDCK) II cells. Here we examined whether EGF affects the stability and intracellular distribution of CLDN2 protein. EGF decreased surface and total levels of CLDN2, which was inhibited by U0126, a MEK inhibitor. CLDN2 was co-localized at the tight junctions (TJs) with ZO-1, a scaffolding protein. The fluorescence signal for CLDN2 disappeared on treatment with EGF, which was inhibited by U0126. EGF accelerated the decrease in CLDN2 in the presence of cycloheximide, a translation inhibitor, indicating that EGF reduces the stability of the protein. Chloroquine, a lysosomal protease inhibitor, blocked the EGF-induced decrease in CLDN2 protein and caused the co-localization of CLDN2 with Lamp-1, a marker of lysosome. Monodancylcadaverine, an inhibitor of endocytosis, and clathrin siRNA blocked the EGF-induced decrease in CLDN2 and the translocation of CLDN2 from the TJs to the lysosome. EGF increased the association of CLDN2 with clathrin and adaptin α which was inhibited by U0126. These results suggest that EGF accelerates clathrin-dependent endocytosis and lysosomal degradation of CLDN2 protein mediated by the activation of a MEK/ERK pathway.     

Phosphatidylinositol-4-Phosphate 5-Kinases and Phosphatidylinositol 4,5-Bisphosphate Synthesis in the Brain

The predominant pathway for phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P₂) synthesis is thought to be phosphorylation of phosphatidylinositol 4-phosphate at the 5 position of the inositol ring by type I phosphatidylinositol phosphate kinases (PIPK): PIPKIα, PIPKIβ, and PIPKIγ. PIPKIγ has been shown to play a role in PI(4,5)P₂ synthesis in brain, and the absence of PIPKIγ is incompatible with postnatal life. Conversely, mice lacking PIPKIα or PIPKIβ (isoforms are referred to according to the nomenclature of human PIPKIs) live to adulthood, although functional effects in specific cell types are observed. To determine the contribution of PIPKIα and PIPKIβ to PI(4,5)P₂ synthesis in brain, we investigated the impact of disrupting multiple PIPKI genes. Our results show that a single allele of PIPKIγ, in the absence of both PIPKIα and PIPKIβ, can support life to adulthood. In addition, PIPKIα alone, but not PIPKIβ alone, can support prenatal development, indicating an essential and partially overlapping function of PIPKIα and PIPKIγ during embryogenesis. This is consistent with early embryonic expression of PIPKIα and PIPKIγ but not of PIPKIβ. PIPKIβ expression in brain correlates with neuronal differentiation. The absence of PIPKIβ does not impact embryonic development in the PIPKIγ knock-out (KO) background but worsens the early postnatal phenotype of the PIPKIγ KO (death occurs within minutes rather than hours). Analysis of PIP₂ in brain reveals that only the absence of PIPKIγ significantly impacts its levels. Collectively, our results provide new evidence for the dominant importance of PIPKIγ in mammals and imply that PIPKIα and PIPKIβ function in the generation of specific PI(4,5)P₂ pools that, at least in brain, do not have a major impact on overall PI(4,5)P₂ levels.     

Genetic Population Structure of Chum Salmon in the Pacific Rim Inferred from Mitochondrial DNA Sequence Variation

We examined the genetic population structure of chum salmon, Oncorhynchus keta, in the Pacific Rim using mitochondrial (mt) DNA analysis. Nucleotide sequence analysis of about 500 bp in the variable portion of the 5′ end of the mtDNA control region revealed 20 variable nucleotide sites, which defined 30 haplotypes of three genealogical clades (A, B, and C), in more than 2,100 individuals of 48 populations from Japan (16), Korea (1), Russia (10), and North America (21 from Alaska, British Columbia, and Washington). The observed haplotypes were mostly associated with geographic regions, in that clade A and C haplotypes characterized Asian populations and clade B haplotypes distinguished North American populations. The haplotype diversity was highest in the Japanese populations, suggesting a greater genetic variation in the populations of Japan than those of Russia and North America. The analysis of molecular variance and contingency χ² tests demonstrated strong structuring among the three geographic groups of populations and weak to moderate structuring within Japanese and North American populations. These results suggest that the observed geographic pattern might be influenced primarily by historic expansions or colonizations and secondarily by low or restricted gene flow between local groups within regions. In addition to the analysis of population structure, mtDNA data may be useful for constructing a baseline for stock identification of mixed populations of high seas chum salmon.     

Noncovalent Immobilization of Streptavidin on In Vitro- and In Vivo-Biotinylated Bacterial Magnetic Particles

Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.     

Arabidopsis HDA6 is required for freezing tolerance

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.