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The soybean cyst nematode (SCN) is one of the most damaging pests affecting soybean production. SCN displays important host recognition behaviors, such as hatching and infection, by recognizing several compounds produced by the host. Therefore, controlling SCN behaviors such as chemotaxis and thermotaxis is an attractive pest control strategy. In this study, we found that cyclic nucleotide-gated channels (CNG channels) regulate SCN chemotaxis and thermotaxis and Hg-tax-2, a gene encoding a CNG channel, is an important regulator of SCN behavior. Gene silencing of Hg-tax-2 and treatment with a CNG channel inhibitor reduced the attraction of second-stage juveniles to nitrate, an attractant with a different recognition mechanism from the host-derived chemoattractant(s), and to host soybean roots, as well as their avoidance behavior toward high temperatures. Co-treatment of ds Hg-tax-2 with the CNG channel inhibitor indicated that Hg-tax-2 is a major regulator of SCN chemotaxis and thermotaxis. These results suggest new avenues for research on control of SCN., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is a neurodegenerative disease characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. Oxidative stress-induced neuronal death has been identified as one of the major causes of nigrostriatal degeneration in PD. The fruit of Actinidia arguta (A. arguta), known as sarunashi in Japan, has been reported to show beneficial health effects such as antioxidant, anti-inflammatory, anti-mutagenic, and anticholinergic effects. In this study, we investigated the neuroprotective effects of A. arguta in 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP)-induced PD model mice. A. arguta juice was administered to 7-week-old C57BL/6J mice continuously for 10 days before the first MPTP injection. The degeneration of dopaminergic neurons in the substantia nigra was induced by MPTP (30 mg/kg, i. p.) once daily for five consecutive days. We found that the administration of A. arguta ameliorated MPTP-induced motor impairment and suppressed the MPTP-induced reductions of tyrosine hydroxylase-positive neurons and tyrosine hydroxylase protein expression in the substantia nigra. Our findings suggest that taking A. arguta could provide neuroprotection that delays or prevents the neurodegenerative process of PD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Eukaryotic proteasome assembly is assisted by multiple dedicated chaperones. In yeast, formation of the heteroheptameric ring composed of α1-α7 subunits is promoted by the heterodimeric chaperone Pba3-Pba4. Here we reveal that in the absence of this dimeric chaperone, α2 replaces α4 during α-ring assembly, thereby giving rise to a non-productive complex that lacks α4, β1, β5, β6, and β7 subunits and aggregates of α4. Furthermore, our structure-guided mutational data demonstrate that the Pba3-Pba4 heterodimer acts as molecular matchmaker reinforcing the interaction between α4 and α5, which is the crucial step in the α-ring formation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures that typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Insulin receptor substrates (IRSs) play central roles in insulin/insulin-like growth factor (IGF) signaling and mediate a variety of their bioactivities. IRSs are tyrosine-phosphorylated by activated insulin receptor/IGF-I receptor tyrosine kinase in response to insulin/IGF, and are recognized by signaling molecules possessing the SH2 domain such as phosphatidylinositol 3-kinase (PI3K), leading to the activation of downstream pathways. Recent studies have suggested that degradation of IRSs by the ubiquitin-proteasome pathway leads to impaired insulin/IGF signaling, but the precise mechanism underlying the process is still unclear. In this study, we identified deubiquitinating enzyme ubiquitin specific protease 7 (USP7) as an IRS-2-interacting protein and demonstrated that deubiquitinase activity of USP7 plays important roles in IRS-2 stabilization through the ubiquitin-proteasome pathway. In addition, insulin treatment dissociated USP7 from IRS-2, leading to degradation of IRS-2. This dissociation was prevented by treatment with LY294002, a PI3K inhibitor, indicating that insulin activation of the PI3K pathway leads to dissociation of IRS-2 from USP7 and IRS-2 degradation. We obtained similar results for IRS-1 in cells treated with insulin and for IRS-2 in cells treated with IGF-I. Taken together, this is the first report demonstrating that USP7 is an IRS-1/2 deubiquitinating enzyme forming a negative feedback loop in insulin/IGF signaling., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The 26S proteasome is a highly conserved multisubunit protease that degrades ubiquitinated proteins in eukaryotic cells. It comprises a 20S core particle and two 19S regulatory particles that are further divided into the lid and base complexes. The lid is a nine subunits complex that is structurally related to the COP9 signalosome and the eukaryotic initiation factor 3. Although the assembly pathway of the 20S and the base are well described, that of the lid is still unclear. In this study, we dissected the lid assembly using yeast lid mutant cells, rpn7-3, Delta rpn9, and rpn12-1. Using mass spectrometry, we identified a number of lid subassemblies, such as Rpn3-Rpn7 pair and a lid-like complex lacking Rpn12, in the mutants. Our analysis suggests that the assembly of the lid is a highly ordered and multi-step process; first, Rpn5, 6, 8, 9, and 11 are assembled to form a core module, then a second module, consisting of Rpn3, 7, and Sem1, is attached, followed by the incorporation of Rpn12 to form the lid complex., ((c) 2010 Elsevier Inc. All rights reserved.)

Saccharomyces cerevisiae Ufd2 is a ubiquitin chain elongation factor in the ubiquitin fusion degradation (UFD) pathway and functions in stress tolerance. A recent study has suggested that the mammalian Ufd2 homologue UFD2a catalyzes formation of Lys27- and Lys33-linked polyubiquitin chains rather than the Lys48-linked chain, but the linkage type of the polyubiquitin chain formed by yeast Ufd2 remains unclear. To determine the property of Ufd2, we reconstituted the UFD pathway using purified enzymes from yeast. Direct determination of the ubiquitin chain linkage type in polyubiquitinated UFD substrates by MALDI-TOF mass spectrometry revealed that Ufd2 catalyzes elongation of the ubiquitin chain through Lys48 linkage.

We examined the role of osteopontin (OPN) in the osteoclastogenesis of arthritis using collagen-induced arthritis (CIA). Cells from arthritic joints of wild-type (OPN +/+) mice spontaneously developed bone-resorbing osteoclast-like cells (OCLs). The cultured cells showed an enhanced expression of receptor activator of nuclear factor kappaB ligand (RANKL) and a decreased expression of osteoprotegerin (OPG). The addition of OPG reduced the number of OCLs, indicating that the osteoclastogenesis depends on the RANK/RANKL/OPG system. The cells also produced OPN abundantly and anti-OPN neutralizing antibodies suppressed the development of OCLs. Moreover, the addition of OPN increased the expression of RANKL and augmented differentiation of OCLs from OPN-deficient (OPN -/-) cells. OPN, like the combination of 1alpha,25-dihydroxyvitamin D(3) and dexamethasone, also enhanced the RANKL expression and decreased OPG expression in a stromal cell line, ST2. These results suggest that OPN acts as a positive regulator in the osteoclastogenesis of arthritis through the RANK/RANKL/OPG system.

To produce a helper virus-free stock of helper-dependent adenoviral vectors (HDAdVs), we have developed a new helper system in which adenoviral genes for propagation of HDAdVs are delivered into producer cells by a herpes simplex virus-1 (HSV) amplicon-adenovirus hybrid. The hybrid amplicon was constructed to carry the E1 gene (HA-E1) or the entire adenoviral genome except E1 (HA-Ad). E1 expression from the HSV amplicon successfully complemented propagation of an E1-deleted adenoviral vector in a human glioma cell line. HDAdVs were propagated in 293 cells infected with HA-Ad. In addition, HDAdVs were rescued and propagated in a glioma cell line superinfected with both HA-E1 and HA-Ad amplicons, although relatively low titers of HSV amplicon resulted in low propagation efficiency of HDAdVs. Since the HSV amplicon can be easily and completely inactivated by chloroform extraction and/or heat treatment from the HDAdV stock, this helper system might be an alternative method to produce helper virus-free HDAdVs.

Ubiquitin-like proteins Rad23 and Dsk2 have recently been shown to be capable of binding both polyubiquitin chains and the 26S proteasome. The ubiquitin-like domains (Ubls) of Rad23 and Dsk2 are indispensable for their interaction with the 26S proteasome, but the proteasome subunits capable of binding the Ubl have not been identified. Here, we report that the Ubls of both Rad23 and Dsk2 can bind with the 19S regulatory particle (RP) of the 26S proteasome in vivo and in vitro. A competition assay using the respective Ubls of Rad23 and Dsk2 revealed that they bind to the RP in a competitive manner. The base subcomplex of the RP was found to have the ability to bind the Ubl. By cross-linking experiments, Rpn1 and Rpn2 were identified as Ubl-binding subunits. Taken together, the results suggest that the Rpn1 and Rpn2 in the base subcomplex form the receptor for the ubiquitin-like protein.

Rpn10, a subunit of the 26S proteasome, has been proposed to act as a receptor for multiubiquitin chains in ubiquitin-dependent proteolysis. However, studies on RPN10-deleted mutants in yeasts have suggested the presence of other multiubiquitin chain-binding factors functioning in ubiquitin-dependent proteolysis. Here, we report that a mutant with a triple deletion of RAD23, DSK2, and RPN10 genes accumulates large amounts of polyubiquitinated proteins, as is the case with a mutant with RAD23 and DSK2 deletions under restrictive conditions. Dsk2, Rad23, and Rpn10 have different capacities to bind multiubiquitin chains. Another ubiquitin-like protein, Ddi1, has similar activity to those of Rad23 and Dsk2. Taken together, the results suggest that ubiquitin-like proteins, Rad23, Dsk2, possibly Ddi1, and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis, serving as multiubiquitin chain-binding proteins., ((c) 2002 Elsevier Science (USA).)

Two sublines of the benzpyrene-induced mouse hepatoma cell line, G-1 and G-5, showed low and high metastatic ability, respectively, to the lung. We produced a polyclonal antibody (pAb) against RAE-1alpha. Five isoforms of RAE-1 have been identified to date, and this pAb recognized all isoforms and was named anti-"pan" RAE-1 pAb. The level of RAE-1 was approximately 5-fold higher in G-5 than in G-1, which was almost RAE-1-negative, as determined using anti-pan RAE-1 pAb. Expression levels of other markers including MHC class I (MHC-I) and Qa-1b were very low and indistinguishable in these sublines. NK-mediated cytotoxicity was determined with these sublines; G-5 was highly susceptible to NK-mediated cytolysis, while G-1 was relatively resistant. The NK-mediated G-5 > G-1 killing profile was diminished if the G-5 cells were pretreated with F(ab)(2)(') of anti-pan RAE-1 pAb. G-1, when transfected with Rae-1alpha cDNA, acquired NK-responsiveness similar to that of G-5. These and additional data using mouse cell lines with low MHC-I levels and various RAE-1 levels also demonstrated that RAE-1 level is critically associated with NK-susceptibility in tumor cells., ((c)2002 Elsevier Science.)

The 26S proteasome, which catalyzes degradation of ubiquitinated proteins, is composed of the 20S proteasome and the 19S complex. Recently, it has been reported that the 26S complex can be dissociated into the lid complex and the 20S-proteasome-base complex in a mutant yeast and that the lid complex is required for ubiquitin-dependent proteolysis. In the present study, we established methods for rapid isolation of the 19S complex, the lid complex, and the base complex from wild-type yeast. The isolated 19S complex was capable of binding to the 20S proteasome to reconstitute the 26S proteasome. In contrast with the previously reported result showing that Rpn10, a multiubiquitin chain binding subunit, is a component of the base complex, we present evidence that the lid complex isolated from wild-type yeast contains Rpn10., (Copyright 2000 Academic Press.)

Previously, we demonstrated the presence of at least two distinct subpopulations of patients with rheumatoid arthritis (RA) employing a cell-transfer experiment using severe combined immunodeficient (SCID) mice. One group of patients, whose T cells derived from the rheumatoid joints, induced synovial hyperplasia (SH) in the SCID mice (the positive group). The other group did not display the induction of SH (the negative group). TCR/Vbeta gene usage analysis indicated that some dominant T cell subpopulations were oligoclonally expanding only in the rheumatoid joints, and not in the periphery of the patients of the positive group. Moreover, these T cell subpopulations were not seen in the joints of patients in the negative group or in non-RA patients. In addition, the preferential uses of certain TCR/Vbetas (Vbeta8, Vbeta12, Vbeta13, and Vbeta14) genes were demonstrated in these T cells. In this study, to investigate whether these T cells are driven by a certain antigen(s), the third complementarity determining regions (CDR3s) of TCR/Vbeta, especially Vbeta8 and Vbeta14 PCR products, were cloned and sequenced. As a result, a dominant CDR3 sequence, CASS-PRERAT-YEQ, was found in Vbeta14+ T cells from the rheumatoid joint of a patient (Patient 1) of the positive group with a Vbeta14 skew. The identical CDR3 sequence also predominated in Vbeta14+ T cells from the rheumatoid joint of another patient (Patient 7) of the positive group with a Vbeta14 skew. In addition, in the patients (Patients 4, 7, 8) of the positive group with a Vbeta8 skew, other dominant CDR3 sequences, CASS-ENS-YEQ and CASS-LTEP-DTQ, were found as in the case of Vbeta14. However, no identical CDR3 sequences were detected dominantly in the joints of the patients in the negative group or in non-RA patients. A Vbeta14+ T cell clone (TCL), named G3, with the identical CDR3 sequence, CASS-PRERAT-YEQ, was isolated successfully from Patient 1, and cell transfer of G3 with autologous irradiated peripheral mononuclear cells induced SH in the SCID mice. Taken together, these results suggest that T cells inducing SH, thought to be pathogenic for RA, might be driven by a certain shared antigen(s)., (Copyright 1999 Academic Press.)

The ribosome-associated protein LBP/p40, which was originally named after "laminin binding protein precursor p40," is distributed on the cell surface as laminin binding protein p67 (LBP/p67), in the nucleus, and on 40S ribosomes. In a broad range of eukaryotes, the localization of LBP/p40 on the 40S ribosome is well conserved. Two yeast homologs of LBP/p40 are believed to be essential for cell viability and each gene product probably corresponds to the assembly and/or stability of the 40S ribosomal subunit. The precise role of LBP/p40 in translation, however, remains to be elucidated, especially in higher eukaryotes. In this report, we used a yeast two-hybrid screening method to isolate molecules associated with human LBP/p40 protein on ribosomes. We found that the 40S ribosomal protein S21 was tightly bound with LBP/p40 in this yeast two-hybrid system and in in vitro analysis. Further, we discovered that the association required a broad region of the LBP/p40 amino acid sequence, which corresponds to the highly conserved region of LBP/p40 homologs among eukaryotes., (Copyright 1999 Academic Press.)

Laminin binding protein precursor p40 (LBP-p40) was long believed to be located exclusively in the cytoplasm. We recently reported localization of epitope-tagged LBP-p40 to the nucleus tightly associated with nuclear structure as well as on ribosomes. In this paper, we analyze the interaction of LBP-p40 with DNA and nuclear proteins in vitro. LBP-p40 was found to bind to a double-stranded DNA cellulose column at moderate salt. However, when mixed with a high salt nuclear extract, LBP-p40 was eluted from the DNA cellulose column only at higher salt. An LBP-p40 affinity column indicated that both histone H1 and in particular the core histones associate with LBP-p40. Using recombinant core histone molecules fused with glutathione S-transferase (GST), we demonstrate that histones H2A, H2B, and H4 are capable of interacting with LBP-p40, whereas H3 is not. These results suggest that association of LBP-p40 with histones H2A, H2B, and H4 confers tight binding of LBP-p40 to chromatin DNA in the nucleus., (Copyright 1998 Academic Press.)

The 26S proteasome is assembled from the 20S proteasome and the regulatory subunit complex in an ATP-dependent manner. In the present study, we found that the ATP-dependent activity and the protein amount of the 26S proteasome change during growth of the budding yeast Saccharomyces cerevisiae. Both levels in the stationary phase are higher than those in the exponentially growing phase. On the other hand, the levels of the 20S proteasome appear to remain unchanged during growth. These results suggest that the 26S proteasome undergoes a growth-dependent change and that the 26S proteasome plays a role in the survival of yeast cells under starvation conditions., (Copyright 1998 Academic Press.)

We isolated a monoclonal antibody M108, which recognized 40 kDa protein (p40) in the cytoplasm, the perinuclear region in interphase and the perichromosomal region during mitosis. As reported previously, it was revealed from the immunofluorescent observation and the biochemical analyses that the nuclear p40 was associated both with the nuclear envelope and the chromatin DNA in interphase nuclei. In this report, we isolated the p40 from cytoplasmic particles, and identified it by extensive microsequencing as LBP/p40, which was considered to be a precursor of laminin binding protein p67 (LBP/p67). Epitope-tagged LBP/p40 was expressed in cultured cells, and the protein was localized in the nucleus as well as in the cytoplasm. Further analysis showed that the nuclear LBP/p40 was tightly associated with the nuclear structures.

Rat type 1 angiotensin II receptor has two subtypes, namely type 1a and type 1b. The regulation of the expressions of these two subtype receptor mRNAs was studied by using a competitive polymerase chain reaction method. The expression of the type 1a mRNA in the liver was negatively and that of the type 1b mRNA in the adrenal was positively modulated by bilateral nephrectomy. In the ventricle of 16 week old Spontaneously hypertensive rat, the expression level of the AT1b receptor mRNA was higher than that in the ventricle of the age-matched Wistar-Kyoto rat, while the expression levels of the AT1a mRNA in the ventricle were almost similar between the two strains at this age. Although type 1a and type 1b have almost similar functional properties, the expressions of their mRNAs were differentially regulated.

Single-strand conformation polymorphism (SSCP) was analyzed to detect a mutation in the transthyretin (TTR) gene from the mother and son showing polyneuropathy with carpal tunnel syndrome. DNA segments containing TTR coding sequence were amplified by polymerase chain reaction, heat denatured and electrophoresed on a neutral polyacrylamide gel. The single-stranded DNA fragments in the gel were transferred to a nylon membrane and hybridized with biotinylated TTR cDNA probe, followed with chemiluminescent DNA detection. The mobility shift was found in the fragments of exon 3 from the patients' DNA. Sequencing analyses of the exon 3 confirmed a T----G base change, resulting in a Leu 58----Arg substitution. TTR Arg 58 is the first mutant TTR gene that has been detected by SSCP analysis. The rapid and sensitive detection of new mutations at various sites on the TTR gene is hereafter possible by the present method in the facilities for non-radioactive experiments.