Rat choline acetyltransferase of the peripheral type differs from that of the common type in intracellular translocation

Choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine, has been implicated to involve multiple isoforms of ChAT mRNA in several animals. Since these isoforms are mostly non-coding splice variants, only a homologous ChAT protein of about 68 kDa has been shown to be produced in vivo. Recent evidence indicates the existence of a protein coding splice variant of ChAT mRNA, which lacks exons 6-9 of the rat ChAT gene. The encoded protein was designated ChAT of a peripheral type (pChAT), because of its preferential expression in the peripheral nervous system as confirmed by Western blot and immunohistochemistry. However, functional significance of pChAT is unknown. To obtain a clue to this question, we examined a possible difference in intracellular trafficking between pChAT and the well-known ChAT of the common type (cChAT) using green fluorescent protein (GFP) in living human embryonic kidney cells. Confocal laser scanning microscopy revealed that pChAT-GFP was detectable in the cytoplasm but not in the nucleus, whereas cChAT-GFP was found in both cytoplasm and nucleus. Following treatment with leptomycin B, a nuclear export pathway inhibitor, pChAT-GFP became detectable in both cytoplasm and nucleus, indicating that pChAT can be translocated to the nucleus. In contrast, the leptomycin B treatment did not seem to affect the content of intranuclear cChAT-GFP. After incubation with protein kinase C inhibitors, enhanced accumulation of pChAT-GFP but not cChAT-GFP occurred in the nucleus. These results clearly indicate that pChAT varies from cChAT in intracellular transportation, probably reflecting the difference in physiological roles between pChAT and cChAT.     

Protein oxidation during aging of the nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans has proven a robust genetic model for studies of aging, including the roles of oxidative stress and protein damage. In this review, we focus on the genetics of select long-lived (e.g., age-1, daf-2, daf-16) and short-lived (e.g., mev-1) mutants that have proven useful in revealing the relationships that exist among oxidative stress, life span, and protein oxidation. The former are known to control an insulin/IGF-1-like pathway in C. elegans, while the latter affect mitochondrial function.     

Deficiency of Calcium-Independent Phospholipase A2 Beta Induces Brain Iron Accumulation through Upregulation of Divalent Metal Transporter 1

Mutations in PLA2G6 have been proposed to be the cause of neurodegeneration with brain iron accumulation type 2. The present study aimed to clarify the mechanism underlying brain iron accumulation during the deficiency of calcium-independent phospholipase A2 beta (iPLA₂β), which is encoded by the PLA2G6 gene. Perl’s staining with diaminobenzidine enhancement was used to visualize brain iron accumulation. Western blotting was used to investigate the expression of molecules involved in iron homeostasis, including divalent metal transporter 1 (DMT1) and iron regulatory proteins (IRP1 and 2), in the brains of iPLA₂β-knockout (KO) mice as well as in PLA2G6-knockdown (KD) SH-SY5Y human neuroblastoma cells. Furthermore, mitochondrial functions such as ATP production were examined. We have discovered for the first time that marked iron deposition was observed in the brains of iPLA₂β-KO mice since the early clinical stages. DMT1 and IRP2 were markedly upregulated in all examined brain regions of aged iPLA₂β-KO mice compared to age-matched wild-type control mice. Moreover, peroxidized lipids were increased in the brains of iPLA₂β-KO mice. DMT1 and IRPs were significantly upregulated in PLA2G6-KD cells compared with cells treated with negative control siRNA. Degeneration of the mitochondrial inner membrane and decrease of ATP production were observed in PLA2G6-KD cells. These results suggest that the genetic ablation of iPLA₂β increased iron uptake in the brain through the activation of IRP2 and upregulation of DMT1, which may be associated with mitochondrial dysfunction.     

Direct binding of RalA to PKCη and its crucial role in morphological change during keratinocyte differentiation

During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) η is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKCη, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKCη, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKCη overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.     

Trophinin-mediated cell adhesion induces apoptosis of human endometrial epithelial cells through PKC-δ

Trophinin is an intrinsic membrane protein expressed in trophectoderm cells of embryos and in uterine epithelial cells. Trophinin potentially mediates apical cell adhesion at human embryo implantation sites through trophinin-trophinin binding in these two cell types. Trophinin-mediated cell adhesion activates trophectoderm cells for invasion, whereas the effect of adhesion on maternal side is not known. We show that addition of GWRQ peptide, a previously established peptide that mimics trophinin-mediated cell adhesion, to human endometrial epithelial cells expressing trophinin induces their apoptosis. FAS involvement was excluded, as GWRQ did not bind to FAS, and FAS knockdown did not alter GWRQ-induced apoptosis. Immunoblotting analyses of protein kinases revealed an elevation of PKC-δ protein in GWRQ-bound endometrial epithelial cells. In the absence of GWRQ, PKC-δ associated with trophinin and remained cytoplasmic, but after GWRQ binding to the trophinin extracellular domain, PKC-δ became tyrosine phosphorylated, dissociated from trophinin and entered the nucleus. In PKC-δ knockdown endometrial cells, GWRQ did not induce apoptosis. These results suggest that trophinin-mediated cell adhesion functions as a molecular switch to induce apoptosis through the PKC-δ pathway in endometrial epithelial cells. Thus, trophinin-mediated induction of apoptosis of endometrial epithelial cells, which function as a barrier to embryo invasion, allows trophoblast invasion of maternal tissue and embryo implantation in humans.Key words: blastocyst, embryo implantation, apoptosis, cell adhesion, signal transduction     

Optical characteristics of flowing blood

The transmitted light strength (TS) through a thin blood layer changes with variation in blood flow, such as positive streaming transparency for low hematocrits and negative streaming transparency for high hematocrits. These phenomena are examined theoretically and experimentally. Maxwell’s equations are solved assuming that erythrocytes are oblate spheroids to investigate these phenomena due to flowing blood. The theoretical results reveal that the scattering and absorption cross sections for flowing blood are larger than those for stagnant blood. Experimental results indicate that the TS for both oxygenated and deoxygenated flowing blood, with a hematocrit of up to approximately 20%, was stronger than that for stagnant blood. The TS decreased for flowing blood with a hematocrit of approximately 20% or greater. Applying the theoretical scattering and absorption cross sections to the absorption and multiple scattering theory of Victor Twersky, the changes in the TS due to flowing blood are obtained theoretically. From the theoretical and experimental results, the positive streaming transparency phenomenon of flowing blood with a low hematocrit and the negative streaming transparency phenomenon with a high hematocrit are found to result from increased scattering and absorption cross sections because of the orientation of flowing erythrocytes.     

Mitochondrial superoxide anion (O(2)(-)) inducible "mev-1" animal models for aging research

Most intracellular reactive oxygen species (ROS), especially superoxide anion (O(2)(-)) that is converted from oxygen, are overproduced by excessive electron leakage from the mitochondrial respiratory chain. Intracellular oxidative stress that damages cellular components can contribute to lifestyle-related diseases such as diabetes and arteriosclerosis, and age-related diseases such as cancer and neuronal degenerative diseases. We have previously demonstrated that the excessive mitochondrial O(2)(-) production caused by SDHC mutations (G71E in C. elegans, I71E in Drosophila and V69E in mouse) results in premature death in C. elegans and Drosophila, cancer in mouse embryonic fibroblast cells and infertility in transgenic mice. SDHC is a subunit of mitochondrial complex II. In humans, it has been reported that mutations in SDHB, SDHC or SDHD often result in inherited head and neck paragangliomas (PGLs). Recently, we established Tet-mev-1 conditional transgenic mice using our uniquely developed Tet-On/Off system, which equilibrates transgene expression to endogenous levels. These mice experienced mitochondrial respiratory chain dysfunction that resulted in O(2)(-) overproduction. The mitochondrial oxidative stress caused excessive apoptosis leading to low birth weight and growth retardation in the neonatal developmental phase in Tet-mev-1 mice. Here, we briefly describe the relationships between mitochondrial O(2)(-) and aging phenomena in mev-1 animal models. [BMB reports 2011; 44(5): 298-305].     

Activation of Imd pathway in hemocyte confers infection resistance through humoral response in Drosophila

Upon microbial invasion the innate immune system of Drosophila melanogaster mounts a response that comes in two distinct but complimentary forms, humoral and cellular. A screen to find genes capable of conferring resistance to the Gram-positive Staphylococcus aureus upon ectopic expression in immune response tissues uncovered imd gene. This resistance was not dependent on cellular defenses but rather likely a result of upregulation of the humoral response through increased expression of antimicrobial peptides, including a Toll pathway reporter gene drosomycin. Taken together it appears that Imd pathway is capable of playing a role in resistance to the Gram-positive S. aureus, counter to notions of traditional roles of the Imd pathway thought largely to responsible for resistance to Gram-negative bacteria.