Novel phosphorelay-dependent control of ZFP36L1 protein during the cell cycle.

The ZFP36 family is a prototypical member of a highly conserved group of proteins with CCCH-type RNA-binding domains, whose functional role and regulatory mechanism in mitotic cells remain obscure. In this study, we provide the first evidence that ZFP36L1 phosphorylation is modulated in a cell cycle-dependent manner. The C-terminal region of ZFP36L1 is critical for its cell cycle-dependent phosphorylation of this protein. We also suggest that the phosphorelay-dependent regulation of ZFP36L1 influences mitotic spindle organization. Thus, our data demonstrate a new class of regulatory mechanism for CCCH-type zinc-finger proteins in cell cycle control.

A conserved island of BAG6/Scythe is related to ubiquitin domains and participates in short hydrophobicity recognition.

BAG6 (also called Scythe) interacts with the exposed hydrophobic regions of newly synthesized proteins and escorts them to the degradation machinery through mechanisms that remain to be elucidated. In this study, we provide evidence that BAG6 physically interacts with the model defective protein substrate CL1 in a manner that depends directly on its short hydrophobicity. We found that the N terminus of BAG6 contains an evolutionarily conserved island tentatively designated the BAG6 ubiquitin-linked domain. Partial deletion of this domain in the BAG6 N-terminal fragment abolished in cell recognition of polyubiquitinated polypeptides as well as the hydrophobicity-mediated recognition of the CL1 degron in cell and in vitro. These observations suggest a mechanism whereby the BAG6 ubiquitin-linked domain provides a platform for discriminating substrates with shorter hydrophobicity stretches as a signal for defective proteins.

A novel signaling pathway mediated by the nuclear targeting of C-terminal fragments of mammalian Patched 1.

BACKGROUND: Patched 1 (Ptc1) is a polytopic receptor protein that is essential for growth and differentiation. Its extracellular domains accept its ligand, Sonic Hedgehog, while the function of its C-terminal intracellular domain is largely obscure. PRINCIPAL FINDINGS: In this study, we stably expressed human Ptc1 protein in HeLa cells and found that it is subjected to proteolytic cleavage at the C-terminus, resulting in the generation of soluble C-terminal fragments. These fragments accumulated in the nucleus, while the N-terminal region of Ptc1 remained in the cytoplasmic membrane fractions. Using an anti-Ptc1 C-terminal domain antibody, we provide conclusive evidence that C-terminal fragments of endogenous Ptc1 accumulate in the nucleus of C3H10T1/2 cells. Similar nuclear accumulation of endogenous C-terminal fragments was observed not only in C3H10T1/2 cells but also in mouse embryonic primary cells. Importantly, the C-terminal fragments of Ptc1 modulate transcriptional activity of Gli1. CONCLUSIONS: Although Ptc1 protein was originally thought to be restricted to cell membrane fractions, our findings suggest that its C-terminal fragments can function as an alternative signal transducer that is directly transported to the cell nucleus.

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells.

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356-5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO.

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development.

Proteasomal ubiquitin receptor RPN-10 controls sex determination in Caenorhabditis elegans.

The ubiquitin-binding RPN-10 protein serves as a ubiquitin receptor that delivers client proteins to the 26S proteasome. Although ubiquitin recognition is an essential step for proteasomal destruction, deletion of the rpn-10 gene in yeast does not influence viability, indicating redundancy of the substrate delivery pathway. However, their specificity and biological relevance in higher eukaryotes is still enigmatic. We report herein that knockdown of the rpn-10 gene, but not any other proteasome subunit genes, sexually transforms hermaphrodites to females by eliminating hermaphrodite spermatogenesis in Caenorhabditis elegans. The feminization phenotype induced by deletion of the rpn-10 gene was rescued by knockdown of tra-2, one of sexual fate decision genes promoting female development, and its downstream target tra-1, indicating that the TRA-2-mediated sex determination pathway is crucial for the Delta rpn-10-induced sterile phenotype. Intriguingly, we found that co-knockdown of rpn-10 and functionally related ubiquitin ligase ufd-2 overcomes the germline-musculinizing effect of fem-3(gf). Furthermore, TRA-2 proteins accumulated in rpn-10-defective worms. Our results show that the RPN-10-mediated ubiquitin pathway is indispensable for control of the TRA-2-mediated sex-determining pathway.

OMA-1 is a P granules-associated protein that is required for germline specification in Caenorhabditis elegans embryos.

In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in the differentiation of germ cells during embryonic development. Previously, we and others have identified novel redundant CCCH-type zinc-finger proteins, OMA-1 and OMA-2, that are involved in oocyte maturation. In this study, we report that the cytoplasmic expression level of OMA-1 protein was largely reduced after fertilization. In contrast to its cytoplasmic degradation, OMA-1 was found to accumulate exclusively on P granules in germline blastomeres during embryogenesis. A notable finding is that embryos with partially suppressed oma-1; oma-2 expression showed inappropriate germline specification, including abnormal distributions of PGL-1, MEX-1 and PIE-1 proteins. Thus, our results suggest that oma gene products are novel multifunctional proteins that participate in crucial processes for germline specification during embryonic development.

Unique proteasome subunit Xrpn10c is a specific receptor for the antiapoptotic ubiquitin-like protein Scythe.

The Rpn10 subunit of the 26S proteasome can bind to polyubiquitinoylated and/or ubiquitin-like proteins via ubiquitin-interacting motifs (UIMs). Vertebrate Rpn10 consists of five distinct spliced isoforms, but the specific functions of these variants remain largely unknown. We report here that one of the alternative products of Xenopus Rpn10, named Xrpn10c, functions as a specific receptor for Scythe/BAG-6, which has been reported to regulate Reaper-induced apoptosis. Deletional analyses revealed that Scythe has at least two distinct domains responsible for its binding to Xrpn10c. Conversely, an Xrpn10c has a UIM-independent Scythe-binding site. The forced expression of a Scythe mutant protein lacking Xrpn10c-binding domains in Xenopus embryos induces inappropriate embryonic death, whereas the wild-type Scythe did not show any abnormality. The results indicate that Xrpn10c-binding sites of Scythe act as an essential segment linking the ubiquitin/proteasome machinery to the control of proper embryonic development.

Neuropsychosocial features of very mild Alzheimer's disease (CDR 0.5) and progression to dementia in a community: the Tajiri project.

The borderline condition between normal aging and dementia should be detected to predict further deterioration. The authors cross-sectionally analyzed neuropsychological data, memory complaints, and social activities for community-dwelling older adults. The rate of decline from Clinical Dementia Rating (CDR) 0.5 to dementia during a 3-year interval was also analyzed. Short-term memory rather than long-term memory was found to be sensitive in distinguishing those with CDR 0 from those with CDR 0.5. Relatives' observations of memory decline rather than subjective memory complaints were significantly different. Participants with CDR 0.5 reported fewer problems with social activities than did their relatives. Ten of the 29 CDR 0.5 participants (34.5%) showed cognitive decline, the decliners showing lower scores on short-term memory and orientation at the baseline condition. The neuropsychological data showed CDR 0.5 to be similar to very mild Alzheimer's disease. It would be better if subjective complaints were excluded from the criteria of the borderline condition.

Intracellularly inducible, ubiquitin hydrolase-insensitive tandem ubiquitins inhibit the 26S proteasome activity and cell division.

We constructed polyubiquitin derivatives that contain a tandem repeat of ubiquitins and were insensitive to ubiquitin hydrolases. They were designated tandem ubiquitin (tUb) with the number of repeats, such as tUb2. When tUbs were expressed under the control of the GAL1 promoter in the wild-type yeast strain, growth was strongly inhibited. Under these conditions, the degradation of N-end rule substrates, a UFD substrate and Gcn4 was inhibited, indicating that the tUb inhibits 26S proteasome activity. Consistent with this, tUb binds to the 26S proteasome. We showed that tUb inhibited the in vitro degradation of polyubiquitinylated Sic1 by the 26S proteasome. When tUB6 messenger RNA was injected into Xenopus embryos, cell division was inhibited, suggesting that tUb can be used as a versatile inhibitor of the 26S proteasome.

Regulation of ascidian Rel by its alternative splice variant.

The Rel/NF-kappaB family of transcription factors play key roles in morphogenesis and immune responses. We reported previously that As-rel1 and As-rel2 of the ascidian Halocynthia roretzi are involved in notochord formation. The As-rel1 protein is a typical Rel/NF-kappaB family member, whereas the As-rel2 protein is a novel truncated product of As-rel1 that lacks a nuclear localization signal and the unique C-terminal region. Here, we present conclusive evidence that As-rel1 and As-rel2 are generated from a single gene by alternative splicing. We analyzed the roles of As-rel2 using cells transfected with As-rel1 or As-rel2 or both. As-rel1 was localized in the nucleus and As-rel2 in the cytoplasm when they were transfected individually. In contrast, when they were transfected simultaneously, both were localized in the nucleus because of the association of As-rel2 with As-rel1. In this case, the transcriptional activity of As-rel1 was suppressed by As-rel2. Ascidian IkappaB was found to sequester As-rel1 in the cytoplasm and suppress its transcriptional activity when As-rel1 and IkappaB were transfected simultaneously. In contrast, when As-rel1 and IkappaB were transfected together with As-rel2, As-rel1 was transported into the nucleus and its transcriptional activity was rescued from inhibition by IkappaB, whereas As-rel2 remained localized in the cytoplasm, suggesting IkappaB sequestration in the cytoplasm by As-rel2. From these findings, we conclude that the alternative splice variant, As-rel2, regulates the nuclear localization and transcriptional activity of As-rel1.

[A case of probable dementia with Lewy bodies presenting with geographic mislocation and nurturing syndrome].

A 83-year-old right handed man developed nurturing syndrome and geographical mislocation (misidentification of places) as a result of dementia with Lewy bodies. He showed parkinsonism, fluctuating cognition, repeated falls, systematic delusions (delusional jealousy with vivid feeling of witness), rapid eye movement sleep behavior disorder, and mild dementia. His brain MRI showed atrophy of bilateral temporal tips and amygdala. A FDG-PET showed decrease of glucose metabolism in right frontal lobe and left temporo-parietal areas. He showed constructional disability, frontal lobe dysfunction, mild deterioration of immediate memory, mild anterograde amnesia, and retrograde amnesia for recent events. Aphasia, apraxia, agnosia, confabulation, or deterioration of facial perception was not noted. He could state the genealogy, ages and recent whereabouts of his relatives, and could state the locations and geographical relationships of his neighboring buildings. He insisted that his father's existence, who had died 52 years ago, although he talked about the episode of his death just before. One month after having a dream that his sick-room was in a fictitious branch of our hospital which located in his neighboring temple, he developed a delusion that his ward was actually in the temple. The former disorder seems to correspond to the nurturing syndrome described by Venneri et al. (2000), and the latter one suggested us that his dream was causally involved in the formation of geographical mis-localization. After Ramachandran's explanation for Capgras' syndrome, we hypothesized that mis-arousal of familiarity evoked by visual perception or memory was attributable to the dysfunction of amygdala, and failure of consistency-checking was caused by the dysfunction of the right frontal lobe.

Corpus callosum atrophy, white matter lesions, and frontal executive dysfunction in normal aging and Alzheimer's disease. A community-based study: the Tajiri Project.

BACKGROUND AND OBJECTIVES: Cerebral MRIs of normal aging and Alzheimer's disease (AD) frequently reveal corpus callosum (CC) atrophy, white matter hyperintensity (WMH), and hippocampal atrophy. However, their relationship or the relationship between these findings and cognitive function has not been fully studied. We investigated the relationship between CC atrophy, WMH, and hippocampal atrophy, together with frontal executive dysfunction in both normal aging and AD. METHOD: We examined 170 randomly selected residents from a designated community: 99 Clinical Dementia Rating (CDR) 0 (healthy, control group, HC) participants, 54 CDR 0.5 (very mild AD) patients, and 17 CDR 1 & 2 (probable AD) patients. By means of MRI, WMH and CC atrophy were visually rated. Four portions of the CC and the hippocampal width were measured. A Mini-Mental State Examination and Cognitive Abilities Screening Instrument (CASI) were performed to assess global function. For the frontal function, the CASI subitems of attention and word fluency, letter-based fluency, the Digit Symbol test of the WAIS-R, and Trail Making Tests were performed. RESULTS: Those patients with CDR 1 & 2 had both hippocampal and CC atrophy, whereas the CDR 0.5 patients had only hippocampal atrophy. Frontal executive dysfunction was associated with CC atrophy in both the HC and AD groups. Significant Spearman correlations were noted between CC atrophy and WMH in both groups. The combined effect of CC atrophy and WMH was noted only in the verbal fluency test in the HC group. CONCLUSION: In both groups, CC atrophy was associated with frontal executive dysfunction. The combined effect of CC atrophy and WMH in normal aging was probably due to subclinical ischemic conditions.

cDNA cloning and characterization of an osmotically sensitive TRP channel from ascidian eggs.

The transient receptor potential (TRP) ion channels are thought to be involved in the entry of calcium ion into cells. In this study, we isolated a cDNA clone, HrTRPV, that shows high homology to Caenorhabditis elegans OSM-9, a TRPV subfamily member of the TRP family, from a Halocynthia roretzi fertilized egg cDNA library. We analyzed its properties using HrTRPV-transfected cells. Upon reduction of extracellular osmolarity, the intracellular calcium concentration was found to increase in HrTRPV-transfected cells. This increase in intracellular calcium concentration was dependent on the presence of extracellular calcium ion and was inhibited by treatment with gadolinium ion, a stretch-activated calcium channel blocker. Thus, these results indicate that ascidian egg HrTRPV is an osmotically sensitive TRP channel.

The 26S proteasome Rpn10 gene encoding splicing isoforms: evolutional conservation of the genomic organization in vertebrates.

Recognition of polyubiquitinated substrates by the 26S proteasome is a key step in the selective degradation of various cellular proteins. The Rpn10 subunit of the 26S proteasome can bind polyubiquitin conjugates in vitro. We have previously reported the unique diversity of Rpn10, which differs from other multiple proteasome subunits, and that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated alternative splicing. To determine whether such alternative splicing mechanisms occur in other species, we searched for Rpn10 isoforms in databases and in our original PCR products. Here we report the genomic organization of the Rpn10 gene in lower vertebrates and provide evidence for the competent generation of distinct forms of Rpn10 by alternative splicing through evolution.

Novel family of CCCH-type zinc-finger proteins, MOE-1, -2 and -3, participates in C. elegans oocyte maturation.

BACKGROUND: Oocyte maturation is an important prerequisite for the production of progeny. Although several germ-line mutations have been reported, the precise mechanism by which the last step of oocyte maturation is controlled remains unclear. In Caenorhabditis elegans, CCCH-type zinc-finger proteins have been shown to be involved in germ cell formation, although their involvement in oocyte maturation has not been fully investigated. RESULTS: Using a multiple RNAi technique, we have identified three novel redundant CCCH-type zinc-finger genes, named by us moe-1, -2 (oma-1, -2) and moe-3, as a group related by functions and nucleotide sequence. Although a single RNAi of each moe gene was not effective, double or triple RNAi induced defects in oocyte maturation. We found that each moe transcript was expressed from the distal to proximal region of the gonad, while their corresponding proteins are accumulated exclusively in proximal oocytes, with a close association to germ granules. Although MOE-2 protein is rapidly removed from germ granules after fertilization, we found that MOE-2 associates with the centrosome-peripheral structure in dividing blastomeres. CONCLUSIONS: Our results suggest that moe gene products are unique multifunctional proteins in terms of their redundancy and characteristic behaviour during the course of oocyte maturation. These gene products participate in processes in the final step of the meiotic cell cycle control, a novel function for CCCH-type zinc-finger family proteins thus far discovered.

Prevalence of dementia and dementing diseases in Japan: the Tajiri project.

BACKGROUND: Vascular dementia (VaD) has been considered to be more prevalent than Alzheimer disease in Japan. However, this might be the result of overdiagnosis stemming from some problematic diagnosis of VaD or of the frequent use of magnetic resonance imaging to detect cerebrovascular disease in older adults. OBJECTIVES: We investigated the prevalence of dementia and the ratios of dementing diseases. The effects of different criteria for VaD (DSM-IV, Alzheimer's Disease Diagnostic and Treatment Centers [ADDTC], and National Institute of Neurological Disorders and Stroke and the Association Internationale pour la Recherche et l'Enseignement en Neurosciences [NINDS-AIREN]) were considered. Hippocampal atrophy and vascular contribution to dementia were evaluated using magnetic resonance imaging findings. METHODS: We targeted all residents 65 years and older (n = 3207) in Tajiri, Japan, and examined 1654 (participant group 1). Of these, 564 (participant group 2) were randomly selected, and 497 underwent magnetic resonance imaging and diagnosis of dementing diseases. RESULTS: We found the overall prevalence of dementia to be 8.5% (141/1654) in participant group 1. Of these, 21 (14.9%) had a history of stroke. Of the 113 participants who had a history of stroke independent of dementia, 18.6% (21/113) were demented. For participant group 2 (n = 497), 32 were demented. The ratio among the dementia for probable VaD based on the NINDS-AIREN criteria was 18.8% (6/32), whereas that for ischemic vascular dementia was 31.3% (10/32) according to the ADDTC criteria. CONCLUSION: We confirmed the overall prevalence of dementia in adults 65 years and older to be 8.5%. We found that VaD was not a common disorder according to the NINDS-AIREN criteria. Rather, the condition of possible Alzheimer disease with cerebrovascular disease was more common.

Discrepancy between mental rotation and perspective-taking abilities in normal aging assessed by Piaget's Three-mountain task.

In a cross-sectional study, we examined age-related differences in visuo-spatial ability associated with image rotation, using two variants of Piaget's 'Three-Mountain Task.' The object-mental rotation (OMR) task detects the ability to mentally rotate an image, whereas the subject-mental rotation (SMR) task reveals the ability to mentally change one's perspective. A group of 33 young adults, 26 middle-aged adults, and 31 elderly normal adults were studied. Both tasks revealed age-related differences in performance but a larger difference between middle-aged and elderly group was observed for SMR than OMR performance. Age-related increases in the 'egocentric' type of error were found only on the SMR task. The results suggest that the ability to mentally change one's perspective declines with age, perhaps more than the ability to mentally rotate objects.

Expression of genes for two C-type lectins during budding of the ascidian Polyandrocarpa misakiensis.

Two closely related cDNA fragments, named pTC14-1 and pTC14-2, encoding C-type lectins were cloned from the budding ascidian Polyandrocarpa misakiensis by means of the polymerase chain reaction. The amino acid sequence deduced from pTC14-1 was identical to that of a 14-kDa calcium-dependent galactose-binding lectin, TC-14, that had been purified from this species. Between the two clones, nucleotide sequence similarity was 90%, whilst that of the deduced amino acid sequences was 82%. The cDNA inserts of these clones hybridized weakly with each other. Antisense RNA probes prepared from these clones gave intense hybridization signals on Northern blots of the W strain, but very weak signals on those of the other strains. Therefore, both clones were suggested to originate from the W strain, but from two separate genes, since the base substitution was scattered throughout the entire translated region. The amount of TC14-1 mRNA increased during bud development, and peaked at 36 h after separation of the bud from the parental body wall. At this stage, extracellular matrix containing TC-14 lectin developed in the mesenchymal space around the morphogenetic region of the bud. There was much less TC14-2, than TC14-1 mRNA at every stage of bud development. TC14-1 and TC14-2 mRNAs were detected on Northern blots of RNAs from adults and growing buds, suggesting that these genes can be used as the earliest markers of budding in this species.