A kunitz-type protease inhibitor bikunin disrupts ligand-induced oligomerization of receptors for transforming growth factor (TGF)-beta and subsequently suppresses TGF-beta signalings

We previously found that bikunin (bik), a Kunitz-type protease inhibitor, suppresses transforming growth factor-beta1 (TGF-beta1)-stimulated expression of urokinase-type plasminogen activator (uPA) in human ovarian cancer cells that lack endogenous bik. In the present study, we tried to elucidate the mechanism by which bik also inhibits plasminogen activator inhibitor type-1 (PAI-1) and collagen synthesis using human ovarian cancer cells. Here, we show that (a) there was an enhanced production of both uPA and PAI-1 in HRA cells in response to TGF-beta1; (b) the overexpression of bik in the cells or exogenous bik results in the inhibition of TGF-beta1 signaling as measured by phosphorylation of the downstream signaling effector Smad2, nuclear translocation of Smad3, and production of PAI-1 and collagen; (c) bik neither decreased expression of TGF-beta receptors (TbetaRI and TbetaRII) in either cell types nor altered the specific binding of 125I TGF-beta1 to the cells, indicating that the effects of bik in these cells are not mediated by ligand sequestration; (d) TbetaRI and TbetaRII present on the same cells exclusively form aggregates in TGF-beta1-stimulated cells; (e) co-treatment of TGF-beta1-stimulated cells with bik suppresses TGF-beta1-induced complex formation of TbetaRI and TbetaRII; and (f) a chondroitin-4-sulfate side chain-deleted bik (deglycosylated bik) does not inhibit TGF-beta1 signaling or association of type I/type II receptor. We conclude that glycosylated bik attenuates TGF-beta1-elicited signaling cascades in cells possibly by abrogating the coupling between TbetaRI and TbetaRII and that this probably provides the mechanism for the suppression of uPA and PAI-1 expression.     

Alzheimer's beta-secretase cleaves a glycosyltransferase as a physiological substrate

Alzheimer's beta-secretase (BACE1) is a membrane-bound protease that cleaves the amyloid precursor protein (APP) in the trans-Golgi network, an initial step in the pathogenesis of Alzheimer's disease. Although BACE1 is distributed among various tissues including brain, its physiological substrate other than APP have not been identified. We have recently found that when BACE1 was overexpressed in COS cells together with alpha2,6-sialyltransferase (ST6Gal I), the secretion of ST6Gal I markedly increased, suggesting that BACE1 cleaves ST6Gal I as a physiological substrate. Thus BACE1 is the first identified protease that is responsible for the cleavage and secretion of glycosyltransferases.     

Involvement of proteases in glycosyltransferase secretion: Alzheimer's β-secretase-dependent cleavage and a following processing by an aminopeptidase

Alzheimer's beta-secretase (BACE1) cleaves amyloid precursor protein to produce amyloid beta-peptide, which is a crucial initiation process of the pathogenesis of Alzheimer's disease. We previously found that BACE1 also cleaves a membrane-bound sialyltransferase (ST6Gal I). Here we report that, when the protein A-ST6Gal I fusion protein, or ST6Gal I-derived peptide, was used as an in vitro substrate for BACE1, it cleaved the substrates between Leu(37) and Gln(38). However, a soluble form of ST6Gal I secreted from COS cells started from Glu(41), which was three amino acids shorter than the in vitro product. The results suggested that the BACE1 product was truncated by an aminopeptidase(s) before secretion. The aminopeptidase activity was successfully detected in detergent extracts of Golgi-membrane fraction. Taken together, we concluded that BACE1 initially cleaved ST6Gal I between Leu(37) and Gln(38), and the NH(2)-terminal three amino acids of the yielded product was further trimmed by the aminopeptidase.     

Nucleotide sequence of a rice cDNA similar to a maize NADP-dependent malic enzyme

We have isolated a rice cDNA clone that is homologous to the gene for the maize NADP-dependent malic enzyme (EC 1.1.1.40; NADP-ME). The deduced amino acid sequence coded for by the cDNA indicates a high level of homology to chloroplast type NADP-ME, including a transit peptide with pronounced hydrophobic properties at the amino terminus. Northern blot analysis indicates that the expression of this gene is regulated by external stress such as submergence.     

Biosynthesis and in vitro enzymatic synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine conjugate of α-linolenic acid

The isoleucine conjugate of 12-oxo-phytodienoic acid (OPDA-Ile), a new member of the jasmonate family, was recently identified in Arabidopsis thaliana and might be a signaling molecule in plants. However, the biosynthesis and function of OPDA-Ile remains elusive. This study reports an in vitro enzymatic method for synthesizing OPDA-Ile, which is catalyzed by reactions of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) using isoleucine conjugates of α -linolenic acid (LA-Ile) as the substrate. A. thaliana fed LA-Ile exhibited a marked increase in the OPDA-Ile concentration. LA-Ile was also detected in A. thaliana. Furthermore, stable isotope labelled LA-Ile was incorporated into OPDA-Ile. Thus, OPDA-Ile is biosynthesized via the cyclization of LA-Ile in A. thaliana.     

Low‐frequency conductivity tensor of rat brain tissues inferred from diffusion MRI

Conductivity tensor maps of the rat brain were obtained using diffusion magnetic resonance imaging (MRI). Signal attenuations in the cortex and the corpus callosum were measured using the stimulated echo acquisition mode (STEAM) sequence with b factors up to 6000 s/mm². Our previously published method was improved to infer 3 × 3 conductivity tensor at the low‐frequency limit. The conductivity tensor of the tissue was inferred from the fast component of the diffusion tensor and a fraction of the fast component. The mean conductivity (MC) of the cortex and the corpus callosum was 0.52 and 0.62 S/m, respectively. Diffusion‐weighted images were obtained with b factors up to 4500 s/mm². Conductivity tensor images were calculated from the fast diffusion tensor images. Tissues with highly anisotropic cellular structures, such as the corpus callosum, the internal capsule, and the trigeminal nerve, exhibited high anisotropy in conductivity. The resulting values corresponded to conductivities at the low‐frequency limit because our method assumed electric currents flowing only through extracellular fluid. Bioelectromagnetics 30:489–499, 2009. © 2009 Wiley‐Liss, Inc.     

How have spawning ground investigations of the Japanese eel Anguilla japonica contributed to the stock enhancement?

After Schmidt’s discovery of the spawning area of the Atlantic eels Anguilla anguilla and A. rostrata, the search for the Japanese eel A. japonica began in the Pacific Ocean. In 1991, the spawning area of the Japanese eel was determined to be the western North Pacific. Because of enthusiastic research, eggs and maturing eels have been collected in the Japanese eel. These findings are the first for one of the 19 freshwater eels. The population sizes of the Japanese and Atlantic eels are linearly decreasing. Thus, these eel population sizes are considered outside of safe biological limits, and the current fisheries are not sustainable. Artificial propagation has not yet succeeded for the freshwater eels. Stock assessment and management of the European eel have received increasing attention; however, such assessments and management of the Japanese eel have not yet been seriously considered. This paper is an overview of the results of intensive spawning ground investigations of the Japanese eel and describes how the outcomes of these studies have contributed not only to biological interests but also to stock enhancement. During the past 20 years of expeditions, noticeable findings have only been collected for wild eggs and mature adult specimens in spite of the expenditure of large research grants and the large amounts of time invested. The outcomes throughout an expedition do not necessarily contribute to the development and improvement of artificial breeding techniques and stock enhancement. Thus, eel research should be more focused on the studies related to eel stock management.     

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.Successful cell division requires faithful replication of the genome, and defects in this process can contribute to genomic instability and subsequent malignant transformation (23). A key regulator of the normal cell cycle is the early mitotic inhibitor 1 (EMI1/FBXO5), a zinc finger protein expressed by a variety of adult tissues and especially in proliferating Ki-67-positive cells (39). Studies of the mammalian and Xenopus homologues of EMI1 have shown that it inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex that targets cell cycle-regulated proteins, such as the S- and M-phase cyclins (A and B), securin, and geminin (13, 25, 31). Depletion of EMI1 by small interfering RNA (siRNA) knockdown in cell lines or immunodepletion in cycling Xenopus extracts results in the untimely degradation of APC/C substrates, delaying G₁/S- and M-phase progression and inducing rereplication (6, 21, 25, 31). Such rereplication is a consequence of decreased levels of the APC/C substrates cyclin A and geminin, which are regulators of replication licensing (6, 21). The result of EMI1 depletion in some cell lines is senescence (39).Despite these insights into the molecular underpinnings of EMI1 function, little is known about the role of this protein in development. Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined. We sought to close this gap by taking advantage of the zebrafish model system. Zebrafish embryos harboring homozygous mutations of emi1 (emi1^m/m) develop beyond the onset of circulation, providing a unique opportunity to examine the developmental roles of Emi1 in vivo. The zebrafish emi1 mutant (hi2648) line was originally identified by a proviral insertional mutagenesis screen designed to identify genes that are necessary for normal morphological development in embryos (1, 8, 9). Subsequent studies showed that the insertion was located between the first and second exons of the emi1 gene (2). The morphological defects in emi1^m/m embryos at 2 days postfertilization (p.f.) are described in the public access zebrafish model organism database (http://zfin.org). Briefly, abnormalities in emi1^m/m embryos can be identified as early as 20 h p.f. and include slightly smaller heads and a lack of ventral curving of the posterior presomitic mesoderm. By 25 h p.f., the tail is more ventrally curved than in normal embryos, and increased cell death is observed throughout the central nervous system. Mutant embryos have circulating blood cells, although the onset of circulation is delayed. We became interested in this mutant because it harbors defects in the numbers and morphology of granulocytes, an important myeloid cell type within the innate immune system.There is evidence that EMI1 may function in cancer pathogenesis, and a variety of human tumors express this factor very highly, although in some cases this may be a consequence of elevated proliferation rates (11, 18). In fact, the human homologue of emi1 resides within chromosome 6q25, a region often deleted in leukemia, which, together with the cell cycle-regulatory role of EMI1, suggested that this factor may also function as a tumor suppressor whose loss of function could promote genetic instability. Thus, in addition to investigating the role of zebrafish emi1 in zebrafish development, with particular emphasis on hematopoiesis, we examined mammalian cells to identify mechanisms that may be important in EMI1-related malignant transformation and explored a putative tumor suppressor role for this cell cycle-regulatory protein.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.