In vitro protein phosphorylation associated with cellular differentiation of Streptomyces griseus

In vitro phosphorylation reactions with crude cellular extracts revealed that phosphorylation of a 17-kDa protein is associated with the onset of aerial mycelium formation in solid culture (but not submerged spore formation in liquid culture) of Streptomyces griseus. The possible importance of the 17-kDa protein phosphorylation in cellular differentiation was further indicated by inducing aerial mycelium formation in the presence of decoyinine and in studies using certain developmental mutants (relC, afsA, and M-1). It is proposed that the 17-kDa protein may play a role in cellular differentiation of S. griseus via its phosphorylation. Received: 17 July 1997 / Accepted: 20 September 1997     

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

PCaPs,possible regulators of PtdInsP signals on plasma membrane

In plants, Ca²⁺, phosphatidylinositol phosphates (PtdInsPs) and inositol phosphates are major components of intracellular signaling. Several kinds of proteins and enzymes, such as calmodulin (CaM), protein kinase, protein phosphatase, and the Ca²⁺ channel, mediate the signaling. Two new Ca²⁺-binding proteins were identified from Arabidopsis thaliana and named PCaP1 and PCaP2 [plasma membrane (PM)-associated Ca²⁺(cation)-binding protein 1 and 2]. PCaP1 has an intrinsically disordered region in the central and C-terminal parts. The PCaP1 gene is expressed in most tissues and the PCaP2 gene is expressed predominantly in root hairs and pollen tubes. We recently demonstrated that these proteins are N-myristoylated, stably anchored in the PM, and are bound with phosphatidylinositol phosphates, especially PtdInsP₂s. Here we propose a model for the switching mechanism of Ca²⁺-signaling mediated by PtdInsPs. Ca²⁺ forms a complex with CaM (Ca²⁺-CaM) when there is an increase in the cytosol free Ca²⁺. The binding of PCaPs with Ca²⁺-CaM causes PCaPs to release PtdInsPs. Until the release of PtdInsPs, the signaling is kept in the resting state.Key words: calcium signal, calmodulin, inositol phosphate, intrinsically disordered protein, myristoylation, phosphatidylinositol phosphate, plasma membrane     

Precocious metamorphosis in the juvenile hormone-deficient mutant of the silkworm, Bombyx mori

Insect molting and metamorphosis are intricately governed by two hormones, ecdysteroids and juvenile hormones (JHs). JHs prevent precocious metamorphosis and allow the larva to undergo multiple rounds of molting until it attains the proper size for metamorphosis. In the silkworm, Bombyx mori, several "moltinism" mutations have been identified that exhibit variations in the number of larval molts; however, none of them have been characterized molecularly. Here we report the identification and characterization of the gene responsible for the dimolting (mod) mutant that undergoes precocious metamorphosis with fewer larval-larval molts. We show that the mod mutation results in complete loss of JHs in the larval hemolymph and that the mutant phenotype can be rescued by topical application of a JH analog. We performed positional cloning of mod and found a null mutation in the cytochrome P450 gene CYP15C1 in the mod allele. We also demonstrated that CYP15C1 is specifically expressed in the corpus allatum, an endocrine organ that synthesizes and secretes JHs. Furthermore, a biochemical experiment showed that CYP15C1 epoxidizes farnesoic acid to JH acid in a highly stereospecific manner. Precocious metamorphosis of mod larvae was rescued when the wild-type allele of CYP15C1 was expressed in transgenic mod larvae using the GAL4/UAS system. Our data therefore reveal that CYP15C1 is the gene responsible for the mod mutation and is essential for JH biosynthesis. Remarkably, precocious larval-pupal transition in mod larvae does not occur in the first or second instar, suggesting that authentic epoxidized JHs are not essential in very young larvae of B. mori. Our identification of a JH-deficient mutant in this model insect will lead to a greater understanding of the molecular basis of the hormonal control of development and metamorphosis.     

A Set of Lotus japonicus Gifu x Lotus burttii Recombinant Inbred Lines Facilitates Map-based Cloning and QTL Mapping

Model legumes such as Lotus japonicus have contributed significantly to the understanding of symbiotic nitrogen fixation. This insight is mainly a result of forward genetic screens followed by map-based cloning to identify causal alleles. The L. japonicus ecotype 'Gifu' was used as a common parent for inter-accession crosses to produce F2 mapping populations either with other L. japonicus ecotypes, MG-20 and Funakura, or with the related species L. filicaulis. These populations have all been used for genetic studies but segregation distortion, suppression of recombination, low polymorphism levels, and poor viability have also been observed. More recently, the diploid species L. burttii has been identified as a fertile crossing partner of L. japonicus. To assess its qualities in genetic linkage analysis and to enable quantitative trait locus (QTL) mapping for a wider range of traits in Lotus species, we have generated and genotyped a set of 163 Gifu × L. burttii recombinant inbred lines (RILs). By direct comparisons of RIL and F2 population data, we show that L. burttii is a valid alternative to MG-20 as a Gifu mapping partner. In addition, we demonstrate the utility of the Gifu × L. burttii RILs in QTL mapping by identifying an Nfr1-linked QTL for Sinorhizobium fredii nodulation.     

Autoantibodies against cardiac troponin I are responsible for dilated cardiomyopathy in PD-1-deficient mice

We recently reported that mice deficient in the programmed cell death-1 (PD-1) immunoinhibitory coreceptor develop autoimmune dilated cardiomyopathy (DCM), with production of high-titer autoantibodies against a heart-specific, 30-kDa protein. In this study, we purified the 30-kDa protein from heart extract and identified it as cardiac troponin I (cTnI), encoded by a gene in which mutations can cause familial hypertrophic cardiomyopathy (HCM). Administration of monoclonal antibodies to cTnI induced dilatation and dysfunction of hearts in wild-type mice. Monoclonal antibodies to cTnI stained the surface of cardiomyocytes and augmented the voltage-dependent L-type Ca2+ current of normal cardiomyocytes. These findings suggest that antibodies to cTnI induce heart dysfunction and dilatation by chronic stimulation of Ca2+ influx in cardiomyocytes.     

Cranial neural crest cell migration in cockatiel Nymphicus hollandicus (Aves: Psittaciformes)

Parrots have developed unique jaw muscles in their evolutionary history. The M. pseudomasseter, which completely covers the lateral side of the jugal bar, is regarded as a jaw muscle unique to parrots. In a previous study, I presented a hypothesis on the relevance of modifications in the regulation of cranial neural crest cell (NCC) development to the generation of this novel jaw muscle based on histological analyses (Tokita [2004] J Morphol 259:69-81). In the present study, I investigated distribution and migration patterns of cranial neural crest cells (NCCs) through parrot embryogenesis with immunohistochemical techniques to further understand the role of cranial NCCs in the evolution of the M. pseudomasseter, and to provide new information on the relative plasticity in cranial NCC migration at early stages of avian development. The basic nature of cranial NCC development was mostly conserved between chick and parrot. In both, cranial NCCs migrated from the dorsal tip of the neural tube in a ventral direction. Three major populations were identified in their cranial NCCs. Migration pathways of these cells were almost identical between chick and parrot. The principal difference was seen in the relative timing of cranial NCC migration. In the parrot, cranial NCC migration into the first pharyngeal arch was more advanced than in the chick at early stages of development. Such a temporal shift in cranial NCC migration might influence architectural patterning of parrot jaw muscles that generates new muscle like M. pseudomasseter.     

Amphibian in vitro heart induction: a simple and reliable model for the study of vertebrate cardiac development

Amphibian embryos are an excellent model system for analyzing the mechanisms of vertebrate cardiogenesis. Studies of heart development in Xenopus have, for example, revealed that the inductive interaction of the heart primordia with the adjacent underlying endoderm and dorsal lip starts at the early stages of gastrulation. However, the molecular basis of those early inductive events and the genes expressed during the early phases of heart differentiation remain largely unknown. Amphibian blastula embryos contain pluripotent cells in their ectodermal region, called the "animal cap," which fortunately can be exploited for understanding a variety of organogenesis processes. Despite an enormous potential for analysis, the use of this system in cardiogenesis research has languished due to a lack of information concerning appropriate culture methods. Herein we report conditions for generating an in vitro heart induction system and present evidence from two types of in vivo transplantations, that the cultured heart rudiment can develop and function in the adult organism. It is expected that the fundamental principles established in this model system will provide a versatile research platform for a variety of organ engineering projects, including modifying in vitro organ growth with exogenous components (e.g. various growth factors) and developing methods for preparing tissue for transplantation.