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We herein elucidate the function of SARS-CoV-2derived 5'UTR in the human cells. 5'UTR bound host cellular RNAs were immunoprecipitated by gRNA-dCas13 (targeting luciferase RNA fused to SARS-CoV-2 5'UTR) in HEK293T and A549 cells. The 5'UTR bound RNA extractions were predominantly enriched for regulating lipid metabolism. Overexpression of SARS-CoV-2 5'UTR RNA altered the expression of factors involved in the process of the human Mevalonate pathway. In addition, we found that HMG-CoA reductase inhibitors were shown to suppress SARS-CoV-2 5'UTR-mediated translation activities. In conclusion, we deduce the array of host RNAs interacting with SARS-CoV-2 5'UTR that drives SARS-CoV-2 translation and influences host metabolic pathways., (© 2023. The Author(s).)

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.

Background: Chorea-acanthocytosis (ChAc) is an autosomal recessive hereditary disease characterized by neurodegeneration in the striatum and acanthocytosis that is caused by mutations in the VPS13A gene. There are only few reports that studied clinical status of the obligate carriers of ChAc. Clinical courses with follow-up neuroradiological and neuropsychological evaluations in individuals with ChAc have been rarely reported., Methods: We followed an index patient with ChAc and evaluated the clinical features of the pedigree members. Genetic analyses of VPS13A and genes responsible for other neuroacanthocytotic and neurodegenerative diseases were performed., Conclusions: The index patient was homozygous for a 3889C>T nonsense mutation in the VPS13A gene and presented with a typical ChAc phenotype. Neuropsychological evaluation with brain imaging in the patient over 3 years revealed atrophy and a decrease in blood flow at the basal ganglia and frontal lobe, and impairment in cognitive function reflecting frontal lobe dysfunction in progressive manners. Four out of five heterozygous mutation carriers in the pedigree showed signs or symptoms potentially attributable to a heterozygous VPS13A mutation.

Mutations in vacuolar protein sorting 13A (VPS13A) gene are responsible for chorea-acanthocytosis (ChAc). We previously determined the full-length sequence and exon-intron structure of mouse VPS13A and generated a ChAc model mouse by using the gene targeting technique. In the process, we found diverse 5' and 3' transcript variants. Since ChAc is a rare neurodegenerative disorder, the mouse model should be useful for investigation of ChAc molecular pathogenesis, and the model's brain specific variants of VPS13A will be indispensable in these investigations. In the present study, we investigated mouse VPS13A transcript variants. We found brain-specific variants of mouse VPS13A, which may be involved in the brain-specific pathology of ChAc. In addition, we identified for the first time mouse VPS13C cDNA sequences and brain-specific variants of VPS13C.

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. In this study, we produced an antibody against chorein and examined its protein-level expression and localization in mouse. Immunoblot analysis revealed that chorein was expressed in a gene dose-dependent manner in the VPS13A deletion-mice that we recently developed, which confirms the sensitivity of the antibody. Chorein was highly expressed in testis, kidney, spleen, and brain, and was expressed ubiquitously in various brain regions. Subcellular analysis of the brain showed high levels of chorein in microsomal and synaptosomal fractions. Immunohistochemically, chorein-like immunoreactivity was ubiquitously observed in the brain in the neuronal perinuclear region, cytoplasm and fibers. In testis and kidney, clear cell-specific patterns of chorein-like immunoreactivity were detected. Our findings provide basic information on chorein in vivo and may contribute to taking the first step toward understanding molecular pathogenesis of ChAc.

Chorea-acanthocytosis (ChAc) is a hereditary neurodegenerative disorder caused by loss of function mutations in the VPS13A gene encoding chorein. Recently, using a gene-targeting technique to delete exons 60-61, we produced a ChAc-model mouse that corresponds to a human disease mutation. In this study, a comparative microarray analysis of gene expression in the striatum revealed an increased level of gephyrin gene expression in the ChAc-model mice compared with wild type mice. Since gephyrin is known as a GABA(A) receptor-anchoring protein, we compared the protein-level expression and localization of gephyrin and the GABA(A) receptor alpha1 (GABRA1) and gamma2 (GABRG2) subunits. Gephyrin and GABRG2 immunoreactivities in the striatum and hippocampus of the ChAc-model mice were significantly higher than those in the wild types. Our results suggest that chorein functional loss may lead to a compensatory upregulation of gephyrin and GABRG2 in the pathologic condition in ChAc.

3-Deoxyglucosone (3-DG) is a metabolite of glucose that is thought to lead to the production of advanced glycation end products in diabetes. The previous assay for 3-DG in serum was based on a multi-step protocol, including derivatization, extraction, HPLC separation, and detection. In the current studies, we established a monoclonal antibody that recognizes the 3-DG-derivative, which is generated by the reaction of 3-DG and a 2,3-diamino-benzene derivative. Attachment of a biotin moiety to the 2,3-diamino-benzene ring via a linker allowed development of a highly sensitive chemiluminescent enzyme immunoassay for 3-DG equivalents. Unlike the previous assay, this method does not require extraction of 3-DG derivatives from serum. Treatment of 3-DG in serum with the DAB-link-biotin produced a quinoxaline derivative, which was specifically recognized by the monoclonal antibody. Using this assay, we found that serum 3-DG was higher in streptozotocin-induced diabetic rats than in normal control rats (25+/-5.6 vs. 9.8+/-1.1 microg/L). This simple assay may allow the monitoring of conditions leading to the accumulation of advanced glycation end products and evaluation of the risk of complications in diabetic patients.

A monoclonal antibody (MAb), gp21-34, specifically reactive with human T-lymphotropic virus type-II (HTLV-II) transmembranous envelope glycoprotein (p20E) was developed by immunization with a recombinant protein fused with thioredoxin moiety at the N-terminal portion. This MAb, which belongs to the IgG1 kappa subclass, reacted with HTLV-II infected cell lines (TON-1, C3-44, and Si-IIA) by IFA, but not with HTLV-I infected cell lines (TCL-Kan and MT-2). By Western blot analysis, this MAb reacted with p20E of HTLV-II lysates but not with HTLV-I lysates. Some epitope analyses with synthetic peptides were carried out to look for a plausible linear epitope in the C-terminal region of p20E.

Mutations in the STA gene at the Xq28 locus have been found in patients with X-linked Emery-Dreifuss muscular dystrophy (EDMD). This gene encodes a hitherto unknown protein named 'emerin'. To elucidate the subcellular localization of emerin, we raised two antisera against synthetic peptide fragments predicted from emerin cDNA. Using both antisera, we found positive nuclear membrane staining in skeletal, cardiac and smooth muscles in the normal controls and in patients with neuromuscular diseases other than EDMD. In contrast, a deficiency in immunofluorescent staining of skeletal and cardiac muscle from EDMD patients was observed. A 34 kD protein is immunoreactive with the antisera--the protein is equivalent to that predicted for emerin. Together, our findings suggest the specific deficiency of emerin in the nuclear membrane of muscle cells in patients with EDMD.

We have shown that a heptapeptide which resides in the middle part of vitronectin (VN) is responsible for binding to plasminogen activator inhibitor-1 (PAI-1). A single PAI-1 binding peptide was isolated from human VN after limited proteolysis with protease V8. The amino acid sequence of the fragment corresponded to residues Gly-115-Glu-121 of VN. A murine monoclonal antibody (JYV-1) raised against human VN bond to the same fragment and inhibited binding of PAI-1 to VN. A synthetic peptide (V-115), comprising residues Gly-115-Glu-121 of human VN, competed with VN for both PAI-1 and JYV-1 in a dose-dependent manner. Synthetic peptide V-111 (Ser-111-Glu-121) had a stronger inhibitory effect than V-115 on binding of PAI-1 or JYV-1 to VN. V-111 also inhibited the binding of human PAI-1 to mouse and rabbit VN. The functional half-life of PAI-1 activity was prolonged approximately 2-fold in the presence of V-111 (1 mM). This stabilizing effect of V-111 was equivalent to intact VN, although a 1000-fold higher molar concentration of V-111 over VN was required. These data indicated that VN residues Gly-115-Glu-121 contain a PAI-1 binding site.