Showing papers in "Journal of Biochemistry in 2013"

Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases.

Abstract: Vascular endothelial growth factors (VEGFs) belong to the platelet-derived growth factor supergene family, and they play central roles in the regulation of angiogenesis and lymphangiogenesis. VEGF-A, the major factor for angiogenesis, binds to two tyrosine kinase (TK) receptors, VEGFR-1 (Flt-1) and VEGFR-2 (KDR/Flk-1), and regulates endothelial cell proliferation, migration, vascular permeability, secretion and other endothelial functions. VEGFR-2 exhibits a strong TK activity towards pro-angiogenic signals, whereas the soluble VEGFR-1 (sFlt-1) functions as an endogenous VEGF inhibitor. sFlt-1 is abnormally overexpressed in the placenta of preeclampsia patients, resulting in the major symptoms of the disease due to abnormal trapping of VEGFs. The VEGF-VEGFR system is crucial for tumour angiogenesis, and anti-VEGF-VEGFR molecules are now widely used in the clinical field to treat cancer patients. The efficacy of these molecules in prolonging the overall survival of patients has been established; however, some cancers do not respond well and reduced tumour sensitivity to anti-VEGF signals may occur after long-term treatment. The molecular basis of tumour refractoriness should be determined to improve anti-angiogenic therapy.

Disialic, oligosialic and polysialic acids: distribution, functions and related disease

Abstract: Sialic acids (Sia) are involved in many biological activities and frequently exist as monosialyl residues at the non-reducing terminal end of glycoconjugates. Occasionally, polymerized structures in the form of disialic acid (diSia), oligosialic acid (oligoSia) and polysialic acid (polySia) are also found in glycoconjugates. In particular, polySia, which is an evolutionarily conserved epitope from sea urchin to humans, is one of the most biologically important glycotopes in vertebrates. The biological functions of polySia, especially on neural cell adhesion molecules, have been well studied and an in-depth body of knowledge concerning polySia has been accumulated. However, considerably less attention has been paid to glycoproteins containing di- and oligoSia groups. However, advances in analytical methods for detecting oligo/polymerized structures have allowed the identification and characterization of an increasing number of glycoproteins containing di/oligo/polySia chains in nature. In addition, sophisticated genetic techniques have also helped to elucidate the underlying mechanisms of polySia-mediated activities. In this review, recent advances in the study of the chemical properties, distribution and functions of di-, oligo- and polySia residues on glycoproteins are described.

The heparin-binding growth factor midkine: the biological activities and candidate receptors

Abstract: The heparin-binding growth factor midkine (MK) comprises a family with pleiotrophin/heparin-binding growth-associated molecule. The biological phenomena in which MK is involved can be categorized into five areas: (i) cancer, (ii) inflammation/immunity, (iii) blood pressure, (iv) development and (v) tissue protection. The phenotypes are clear in vivo, but the mechanisms by which MK exerts these actions are not fully understood. Candidate receptors for MK include anaplastic lymphoma kinase, protein tyrosine phosphatase ζ, Notch2, LDL receptor-related protein 1, integrins and proteoglycans. Some physical associations between these candidate receptors are also known. Because of the striking in vivo phenotypes after manipulation of MK, MK could be an important molecular target for the treatment of various diseases. To this end, it will be important to pursue studies to fully understand the mechanisms of MK action.

Generation of membrane diversity by lysophospholipid acyltransferases.

Abstract: Glycerophospholipids are main components of cellular membranes and have numerous structural and functional roles to regulate cellular functions. Polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, are mainly located at the sn-2, but not the sn-1 position of glycerophospholipids in an asymmetrical manner and the fatty acid compositions at both the sn-1 and sn-2 positions differ in various cell types and tissues. Asymmetry and diversity of membrane glycerophospholipids are generated in the remodelling pathway (Lands' cycle), which are conducted by the concerted actions of phospholipases A2 (PLA2s) and lysophospholipid acyltransferases (LPLATs). The Lands' cycle was first reported in the 1950s. While PLA2s have been well characterized, little is known about the LPLATs. Recently, several laboratories, including ours, isolated LPLATs that function in the Lands' cycle from the 1-acylglycerol-3-phosphate O-acyltransferase family and the membrane bound O-acyltransferases family. In this review, we summarize recent studies on cloning and characterization of LPLATs that contribute to membrane asymmetry and diversity.

Immunology and bone

Abstract: It is now well acknowledged that the immune and skeletal systems interact and affect one another during developmental physiology and pathology. With the aid of modern conditional gene targeting and transgenic technologies, this field of interdisciplinary research, known as osteoimmunology, is rapidly advancing. Numerous bone phenotypes have been described in immune-compromised gene-deficient mice and, albeit to a lesser extent, immune deficiencies exist in osteo-compromised gene-deficient mice, suggesting that bone cells themselves actually regulate the development of immune cells directly. In this review, I discuss the essential role of key cytokines, signalling transduction pathways and transcription factors during immune and bone development, and how pathology driven dysregulation of these shared mechanisms can lead to clinical manifestations. Diseases that are within the remit of osteoimmunology continue to cause significant morbidity, for example, rheumatoid arthritis, osteoporosis, multiple myeloma and breast/prostate cancer. The complexity and overlapping cellular and molecular interactions between the immune and bone tissues, mean that despite fervent research of these diseases, it remains a major challenge to discover therapeutics that can specifically target one system without detrimentally affecting the other.

Deregulation of hyaluronan synthesis, degradation and binding promotes breast cancer

Abstract: Clinical and experimental data indicate that hyaluronan accumulates in breast cancer compared with normal breast epithelium, which correlates to poor prognosis. In this review, we discuss the expression of genes encoding enzymes that synthesize or degrade hyaluronan, i.e. hyaluronan synthases and hyaluronidases or bind hyaluronan, i.e. CD44 and receptor for hyaluronan-mediated motility (RHAMM, also designated as HMMR or CD168), in relation to breast cancer progression. Hyaluronan and hyaluronan receptors have multi-faceted roles in signalling events in breast cancer. A better understanding of the molecular mechanisms underlying these signalling pathways is highly warranted and may lead to improvement of cancer treatment.

The vasohibin family: a novel family for angiogenesis regulation.

Abstract: Angiogenesis, a formation of neovessels, is regulated by the local balance between angiogenesis stimulators and inhibitors. A number of such endogenous regulators of angiogenesis have been found in the body. Recently, vasohibin-1 (VASH1) was isolated as a negative feedback regulator of angiogenesis produced by endothelial cells (ECs) and subsequently vasohibin-2 (VASH2) as a homologue of VASH1. It was then explored that VASH1 is expressed in ECs to terminate angiogenesis, whereas VASH2 is expressed in cells other than ECs to promote angiogenesis in the mouse model of angiogenesis. This review will focus on the vasohibin family members, which are novel regulators of angiogenesis.

Regulation of TGF-β family signalling by ubiquitination and deubiquitination.

Abstract: Members of the transforming growth factor-β (TGF-β) family, including TGF-βs, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. TGF-β family signalling is mainly mediated by membranous serine/threonine kinase receptors and intracellular Smad proteins. This signalling is tightly regulated by various post-translational modifications including ubiquitination. Several E3 ubiquitin ligases play a crucial role in the recognition and ubiquitin-dependent degradation of TGF-β family receptors, Smad proteins and their interacted proteins to regulate positively and negatively TGF-β family signalling. In contrast, non-degradative ubiquitin modifications also regulate TGF-β family signalling. Recently, in addition to protein ubiquitination, deubiquitination by deubiquitinating enzymes has been reported to control TGF-β family signalling pathways. Interestingly, more recent studies suggest that TGF-β signalling is not only regulated via ubiquitination and/or deubiquitination, but also it relies on ubiquitination for its effect on other pathways. Thus, ubiquitin modifications play key roles in TGF-β family signal transduction and cross-talk between TGF-β family signalling and other signalling pathways. Here, we review the current understandings of the positive and negative regulatory mechanisms by ubiquitin modifications that control TGF-β family signalling.

Linear ubiquitination-mediated NF-κB regulation and its related disorders

Abstract: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-κB (NF-κB) signalling. NF-κB is crucial for inflammatory and immune responses, and aberrant NF-κB signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-κB pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-κB activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson's disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-κB activation pathway regulation by LUBAC.

Tumour endothelial cells acquire drug resistance in a tumour microenvironment.

Abstract: Tumour growth is dependent on angiogenesis, and tumour blood vessels are recognized as an important target for cancer therapy. Tumour endothelial cells (TECs) are the main targets of anti-angiogenic therapy. Unlike the traditionally held view, some TECs may be genetically abnormal and might acquire drug resistance. Therefore, we investigated the drug resistance of TECs and the mechanism by which it is acquired. TECs show resistance to paclitaxel through greater mRNA expression of multidrug resistance 1, which encodes P-glycoprotein, as compared with normal endothelial cells. We found that high levels of vascular endothelial growth factor in tumour-conditioned medium may be responsible for upregulated P-glycoprotein expression. This is a novel mechanism for the acquisition of drug resistance by TECs in a tumour microenvironment. This review focuses on the possibility that TECs can acquire drug resistance.

Emerging roles of recycling endosomes

Abstract: Cells internalize extracellular solutes, ligands and proteins and lipids in the plasma membrane (PM) by endocytosis. The removal of membrane from the PM is counteracted by endosomal recycling pathways that return the endocytosed proteins and lipids back to the PM. Recycling to the PM can occur from early endosomes. However, many cells have a distinct subpopulation of endosomes that have a mildly acidic pH of 6.5 and are involved in the endosomal recycling. These endosomes are dubbed recycling endosomes (REs). In recent years, studies have begun to reveal that function of REs is not limited to the endosomal recycling. In this review, I summarize the nature of membrane trafficking pathways that pass through REs and the cell biological roles of these pathways.

The CBL-interacting protein kinase CIPK26 is a novel interactor of Arabidopsis NADPH oxidase AtRbohF that negatively modulates its ROS-producing activity in a heterologous expression system.

Abstract: The plant NADPH oxidases, known as respiratory burst oxidase homologues (Rbohs), play an indispensable role in a wide array of cellular and developmental processes. Arabidopsis thaliana RbohF (AtRbohF)-mediated production of reactive oxygen species (ROS) is involved in biotic and abiotic stress responses. Because of the toxicity of excess amount of ROS, the ROS-producing activity of Rbohs is speculated to be negatively regulated. However, its mechanism is mostly unknown to date. Here, we report the identification of calcineurin B-like protein-interacting protein kinase 26 (CIPK26) as a novel regulatory factor of AtRbohF. We isolated CIPK26 as an AtRbohF-interacting partner by a yeast two-hybrid screen. Our co-immunoprecipitation assay revealed that the CIPK26 protein interacts with the N-terminal region of AtRbohF in Nicotiana benthamiana cell extracts. The fluorescence of both GFP-tagged CIPK26 and AtRbohF was predominantly observed at the cell periphery. We also showed that co-expression of CIPK26 decreases the ROS-producing activity of AtRbohF in HEK293T cells. Together, these results suggest that the direct binding of CIPK26 to AtRbohF negatively modulates ROS production and play a role in the regulation of ROS signalling in plants.

BAG6/BAT3: emerging roles in quality control for nascent polypeptides

Abstract: BAG6 (also known as BAT3/Scythe) is a ubiquitin-like protein that is thought to participate in a variety of seemingly unrelated physiological and pathological processes, such as apoptosis, antigen presentation and the T-cell response. Recent studies have shown that BAG6 is essential for the quality control of aggregation-prone polypeptide biogenesis. It forms part of a complex that determines the fate of newly synthesized client proteins for membrane insertion, ubiquitin-mediated degradation and/or aggregate formation. A biologically relevant transmembrane protein family has recently been shown to be a major client of BAG6, suggesting that many of the known diverse BAG6 functions can be interpreted by BAG6-mediated control of membrane protein biogenesis. In this review, we summarize the current understanding of the physiological roles of BAG6 with a particular focus on quality control for nascent chain polypeptides.

Phosphatidylserine recognition and induction of apoptotic cell clearance by Drosophila engulfment receptor Draper.

Abstract: The membrane phospholipid phosphatidylserine is exposed on the cell surface during apoptosis and acts as an eat-me signal in the phagocytosis of apoptotic cells in mammals and nematodes. However, whether this is also true in insects was unclear. When milk fat globule-epidermal growth factor 8, a phosphatidylserine-binding protein of mammals, was ectopically expressed in Drosophila, the level of phagocytosis was reduced, whereas this was not the case for the same protein lacking a domain responsible for the binding to phosphatidylserine. We found that the extracellular region of Draper, an engulfment receptor of Drosophila, binds to phosphatidylserine in an enzyme-linked immunosorbent assay-like solid-phase assay and in an assay for surface plasmon resonance. A portion of Draper containing domains EMI and NIM located close to the N-terminus was required for binding to phosphatidylserine, and a Draper protein lacking this region was not active in Drosophila. Finally, the level of tyrosine-phosphorylated Draper, indicative of the activation of Draper, in a hemocyte-derived cell line was increased after treatment with phosphatidylserine-containing liposome. These results indicated that phosphatidylserine serves as an eat-me signal in the phagocytic removal of apoptotic cells in Drosophila and that Draper is a phosphatidylserine-binding receptor for phagocytosis.

The ubiquitin code and its decoding machinery in the endocytic pathway.

Abstract: The level of individual plasma membrane proteins needs to be regulated strictly depending on the situation under which the cell is placed. To reduce the level of a specific plasma membrane protein in a short period, cells internalize the protein from the cell surface by endocytosis and degrade it in the lysosome. Internalized cargo proteins are transported to the limiting membrane of the early endosome, from which they are incorporated into the lumenal vesicles of the endosome. Such endosomes, called the late endosome or multivesicular body, fuse with the lysosome, thereby delivering cargo proteins to the lysosomal lumen and exposing them to acid hydrolases. During this lysosomal trafficking process, ubiquitination serves as a signal that drives internalization and endosome-to-lysosome transport of the cargo proteins. In this review, we discuss the types of ubiquitination that drive these trafficking processes, and how the ubiquitin (Ub) modifications are recognized by specific Ub-binding proteins.

Stereochemical assignment and anti-inflammatory properties of the omega-3 lipid mediator resolvin E3

Abstract: Uncontrolled inflammation is now considered to be a link between many widely occurring diseases. Thus, controlling the innate inflammatory response and its local chemical mediators has been receiving increasing attention. We recently identified a novel family of eicosapentaenoic acid (EPA)-derived mediators produced by eosinophils, denoted as resolvin E3 (RvE3), that possess potent anti-inflammatory actions both in vitro and in vivo. Carbons at 17 and 18 positions are asymmetric and thus the molecule has a total of four potential stereoisomers. Here, we assigned the stereochemistry of the conjugated double bonds and chirality of alcohols present in two natural isomers of RvE3 with four different stereoisomers prepared by total organic synthesis. The complete structures of two natural isomers of RvE3 were determined to be 17R,18S- and 17R,18R-dihydroxy-5Z,8Z,11Z,13E,15E-EPA, respectively. These natural isomers prepared by total organic synthesis displayed a potent anti-inflammatory action by limiting neutrophil infiltrations both in vitro and in vivo. The unnatural stereoisomers were much less active compared with the natural isomers, demonstrating the stereoselective action of RvE3.

Evolutionarily conserved regulation of TOR signalling.

Abstract: The target of rapamycin (TOR) is an evolutionarily conserved protein kinase that regulates cell growth in response to various environmental as well as intracellular cues through the formation of 2 distinct TOR complexes (TORC), TORC1 and TORC2. Dysregulation of TORC1 and TORC2 activity is closely associated with various diseases, including diabetes, cancer and neurodegenerative disorders. Over the past few years, new regulatory mechanisms of TORC1 and TORC2 activity have been elucidated. Furthermore, recent advances in the study of TOR inhibitors have revealed previously unrecognized cellular functions of TORC1. In this review, we briefly summarize the current understanding of the evolutionarily conserved TOR signalling from upstream regulators to downstream events.

Endoplasmic reticulum aminopeptidases: biochemistry, physiology and pathology.

Abstract: The human endoplasmic reticulum aminopeptidase (ERAP) 1 and 2 proteins were initially identified as homologues of human placental leucine aminopeptidase/insulin-regulated aminopeptidase. They are categorized as a unique class of proteases based on their subcellular localization on the luminal side of the endoplasmic reticulum. ERAPs play an important role in the N-terminal processing of the antigenic precursors that are presented on the major histocompatibility complex (MHC) class I molecules. ERAPs are also implicated in the regulation of a wide variety of physiological phenomena and pathogenic conditions. In this review, the current knowledge on ERAPs is summarized.

A lactoferrin-receptor, intelectin 1, affects uptake, sub-cellular localization and release of immunochemically detectable lactoferrin by intestinal epithelial Caco-2 cells

Abstract: Intelectin 1 (IntL) is known as a lectin expressed in intestinal epithelia and also as a receptor for an iron-binding protein, lactoferrin (LF). Uptake of LF with bound iron by enterocytes via receptor-mediated endocytosis has been well investigated, whereas subsequent fate of endocytized LF and LF/IntL complexes remains largely unknown. In the present study, we examined contribution of IntL to the uptake, sub-cellular localization and subsequent release of LF by intestinal Caco-2 IntL-transfectants using two-site ELISA and fluorescence confocal microscopy. LF taken up by IntL-transfectants was immunochemically detected mostly as intact protein in the cell lysates, and it was a little larger in amount than that of the mock-transfectants. In the IntL-transfectants cultured on porous membrane, LF taken up from the apical side was detected immunochemically as punctate signals in the apical-side cytoplasmic region near nucleus. The LF signals were co-localized with IntL and, in a time-dependent manner, partially with early endosome antigen 1 (EEA1), but not with alkaline phosphatase. LF taken up, retained and subsequently released by the IntL-transfectants was larger in amount than that of mock-transfectants. Moreover, uptake of LF altered sub-cellular localization of IntL and markedly enhanced the IntL signals within the cells.

EMT and EndMT: regulated in similar ways?

Abstract: Epithelial-mesenchymal transition (EMT) is an evolutionary conserved developmental process, which is evoked during tumour invasion and metastasis. In the tumour microenvironment, a variety of resident and recruited cells participate in tumour progression. Kawata et al. demonstrated an experimental model where proinflammatory cytokines derived from macrophages could enhance EMT of cancer cells. Endothelial-mesenchymal transition (EndMT) is originally observed during heart development, and recent studies suggest its role in pathological settings such as cancer and fibrosis. Mihira et al. demonstrated a line of evidence showing endothelial cell plasticity to undergo EndMT in vitro. Both in EMT and EndMT, transforming growth factor-β played pivotal roles, and multiple downstream mechanisms were used, depending on cell context. These recent works unraveled discrete regulatory networks in mesenchymal transition of epithelial and endothelial cells.

Mechanistic insights into the regulation of circular dorsal ruffle formation.

Abstract: Growth factor stimulations induce dynamic changes in the cytoskeleton beneath the plasma membrane. Among them is the formation of membrane ruffles organized in a circular array, called 'circular dorsal ruffles' (CDRs). Physiological functions of CDRs include downregulation of cell growth by desensitizing the signalling from growth factor receptors as well as rearrangement of adhesion sites at the onset of cell migration. For the formation of CDRs, not only the activators of actin polymerization, such as N-WASP and the Arp2/3-complex, but also membrane deforming proteins with BAR/F-BAR domains are necessary. Small GTPases are also involved in the formation of CDRs by controlling intracellular trafficking through endosomes. Moreover, recent analyses of another circular cytoskeletal structure, podosome rosettes, have revealed common molecular features shared with CDRs. Among them, the roles of PI3-kinase and phosphoinositide 5-phosphatase may hold the key to the induction of these circular structures.

Pharmacophore identification of ascofuranone, potent inhibitor of cyanide-insensitive alternative oxidase of Trypanosoma brucei.

Abstract: Trypanosoma brucei is a parasite that causes human African trypanosomiasis (HAT). The parasites depend on the cyanide-insensitive trypanosome alternative oxidase (TAO) for their vital aerobic respiration. Ascofuranone (AF), a potent and specific sub-nanomolar inhibitor of the TAO quinol oxidase, is a potential novel drug with selectivity for HAT, because mammalian hosts lack the enzyme. To elucidate not only the inhibition mechanism but also the inhibitor-enzyme interaction, AF derivatives were designed and synthesized, and the structure-activity relationship was evaluated. Here we identified the pharmacophore of AF that interacts with TAO. The detailed inhibitory profiles indicated that the 1-formyl and 6-hydroxyl groups, which might contribute to intramolecular hydrogen bonding and/or serve as hydrogen-bonding donors, were responsible for direct interaction with the enzyme.

Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

Abstract: Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

Transforming growth factor-β1 induces epithelial-mesenchymal transition and integrin α3β1-mediated cell migration of HSC-4 human squamous cell carcinoma cells through Slug.

Abstract: We investigated whether transforming growth factor (TGF)-β1 promoted epithelial-mesenchymal transition (EMT) and migration of human oral squamous cell carcinoma (hOSCC) cells. Among 6 hOSCC cell lines investigated, Smad2 phosphorylation and TGF-β target genes expression were most clearly upregulated following TGF-β1 stimulation in HSC-4 cells, indicating that HSC-4 cells were the most responsive to TGF-β1. In addition, the expression levels of the mesenchymal markers N-cadherin and vimentin were most clearly induced in HSC-4 cells among the hOSCC cell lines by TGF-β1 stimulation. Interestingly, E-cadherin and β-catenin at the cell surface were internalized in HSC-4 cells stimulated with TGF-β1. In addition, the expression levels of the EMT-related transcription factor Slug was significantly upregulated on TGF-β1 stimulation. Moreover, the downregulation of Slug by RNA interference clearly inhibited the TGF-β1-induced expression of mesenchymal marker and the migration of HSC-4 cells. Proteomics analysis also revealed that the expression levels of integrin α3β1-targeted proteins were upregulated in TGF-β1-stimulated HSC-4 cells. Neutral antibodies against integrin α3 and β1, as well as a focal adhesion kinase (FAK) inhibitor, clearly suppressed TGF-β1-induced cell migration. These results suggest that the EMT and integrin α3β1/FAK pathway-mediated migration of TGF-β1-stimulated HSC-4 hOSCC cells is positively controlled by Slug.

Initiation of meiotic recombination in chromatin structure

Abstract: Meiotic homologous recombination is markedly activated during meiotic prophase to play central roles in faithful chromosome segregation and conferring genetic diversity to gametes. It is initiated by programmed DNA double-strand breaks (DSBs) by the conserved protein Spo11, and preferentially occurs at discrete sites called hotspots. Since the functions of Spo11 are influenced by both of local chromatin at hotspots and higher-order chromosome structures, formation of meiotic DSBs is under regulation of chromatin structure. Therefore, investigating features and roles of meiotic chromatin is crucial to elucidate the in vivo mechanism of meiotic recombination initiation. Recent progress in genome-wide chromatin analyses tremendously improved our understanding on this point, but many critical questions are left unaddressed. In this review, we summarize current knowledge in the field, and also discuss the future problems that must be solved to understand the role of chromatin structure in meiotic recombination.

Accurate determination of tissue steroid hormones, precursors and conjugates in adult male rat.

Abstract: The actual levels of steroid hormones in organs are vital for endocrine, reproductive and neuronal health and disorders. We developed an accurate method to determine the levels of steroid hormones and steroid conjugates in various organs by an efficient preparation using a solid-phase-extraction cartridge. Each steroid was identified by the precursor ion spectra using liquid chromatography–electrospray ionization time-of-flight mass spectrometry, and the respective steroids were quantitatively analysed in the selected reaction monitoring mode by liquid chromatograph-mass spectrometry/mass spectrometry (LC-MS/MS). The data showed that significant levels of testosterone, corticosterone and precursors of both hormones were detected in all organs except liver. The glucuronide conjugates of steroid hormones and the precursors were detected in all organs except liver, but sulfate conjugates of these steroids were observed only in the target organs of the hormones and kidney. Interestingly, these steroids and the conjugates were not observed in the liver except pregnenolone. In conclusion, an accurate determination of tissue steroids was developed using LC-MS analysis. Biosynthesis of steroid hormones from the precursors was estimated even in the target organs, and the delivery of these steroid conjugates was also suggested via the circulation without any significant hepatic participation.

Phosphatidylinositol-3,5-bisphosphate: metabolism and physiological functions

Abstract: Phosphatidylinositol (PtdIns) is a membrane phospholipid composed of diacylglycerol and a D-myo-inositol head group. In mammals, the hydroxyl groups at the D3, D4 and D5 positions of the inositol ring can be phosphorylated to yield seven phosphoinositide derivatives. PtdIns-3,5-bisphosphate [PtdIns(3,5)P2] is the most recently discovered species of phosphoinositide that is generated by the phosphorylation of PtdIns(3)P at the D5 position by PtdIns phosphate kinase and catabolized through the dephosphorylation by myotubularin family of phosphatases. Genetic and biochemical analyses of the enzymes metabolizing PtdIns(3,5)P2 have revealed that this phospholipid is involved in the control of endolysosomal systems and plays crucial roles in various mammalian tissues. In this article, we review the current state of knowledge of the metabolic/physiological functions of PtdIns(3,5)P2, and describe how disruption of these functions may contribute to human diseases.

Molecular mechanisms of syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cells

Abstract: Syndecan-4, a cell-surface heparan sulfate proteoglycan, can participate in inflammation and wound healing as a host defense molecule. Tumour necrosis factor (TNF)-α, one of the most potent proinflammatory cytokines, is known to upregulate syndecan-4 expression, but the precise mechanisms are unclear. To elucidate these mechanisms in detail, we examined syndecan-4 upregulation by TNF-α in the endothelium-like EAhy926 cell. Of the two putative nuclear factor kappa-B (NF-κB) binding sites in the syndecan-4 gene (SDC4) promoter, deletion or mutation of one or both sites significantly diminished the effects of TNF-α. Electrophoretic mobility shift assays showed that p65 and c-Rel, but not p50, bound to these NF-κB binding sites, whereas pull-down assays showed binding of all three NF-κB components. Chromatin immunoprecipitation assays clearly showed that p65 and phosphorylated p65, but not p50 or c-Rel, bound to the SDC4 promoter. An NF-κB inhibitor, p65 knockdown and a transcriptional elongation inhibitor completely blocked the effect of TNF-α on SDC4 promoter activity and significantly, but not completely, blocked that on SDC4 mRNA expression. These data suggest that NF-κB p65 could be a key mediator of syndecan-4 upregulation by TNF-α through two binding sites in the SDC4 promoter, but other NF-κB-p65 independent pathways might also be involved through transcriptional elongation.

X-Ray crystallographic evidence for the presence of the cysteine tryptophylquinone cofactor in l-lysine ε-oxidase from Marinomonas mediterranea

Abstract: We have determined the x-ray crystal structure of L-lysine e-oxidase from Marinomonas mediterranea in its native and L-lysine-complex forms at 1.94- and 1.99-A resolution, respectively. In the native enzyme, electron densities clearly indicate the presence of cysteine tryptophylquinone (CTQ) previously identified in quinohemoprotein amine dehydrogenase. In the L-lysine-complex, an electron density corresponding to the bound L-lysine shows that its e-amino group is attached to the C6 carbonyl group of CTQ, suggesting the formation of a Schiff-base intermediate. Collectively, the present crystal structure provides the first example of an enzyme employing a tryptophylquinone cofactor in an amine oxidase.

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

Abstract: Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.