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Abstract Background An antisense transcript of histone H2a that has no significant protein-coding region has been cloned from a mouse full-length cDNA library. In the present study, we evaluated this transcript by using RT-PCR and compared the expression patterns of the sense and antisense transcripts by using quantitative RT-PCR (qRT-PCR). Results This antisense RNA was expressed in three mouse cell lines. We call it ASH2a. ASH2a includes not only the complementary sequence of the transcript of Hist2h2aa2 (a replication-dependent histone H2a gene), but also that of the promoter of Hist2h2aa2. The upstream genomic sequence of the transcription start site of the ASH2a-coding gene (ASH2a) lacks both CCAAT and TATA boxes. This absence suggests that the regulation of ASH2a is different from that of the replication-dependent histone H2a genes. Findings from qRT-PCR indicated that the expression pattern of ASH2a was different from that of Hist2h2aa2. Expression of Hist2h2aa2 peaked at 2 to 4 h during S-phase, but that of ASH2a peaked at 1 h. Conclusion We showed the existence of ASH2a, a histone H2a antisense RNA, in mouse cultured cells. The expression pattern of ASH2a is different from that of the sense RNA.

Hypokalemic periodic paralysis (HPP) is a rare neuromuscular disease characterized by periodic attacks of acute muscle weakness concomitant with low serum potassium. To date, only one variant in the ATP1A2 , which encodes the α2 subunit of the Na+/K+ ATPase and is abundantly expressed in skeletal muscle and brain astrocytes, has been reported to cause primary HPP along with intellectual disability. The previous case did not present distinct pathological abnormalities. In this report, we describe a case of HPP in a child, highlighting unique pathological findings. Case presentation: The case is a 3-year-old girl. She had weak sucking ability and poor weight gain after birth. She had mild hypotonia, motor developmental delay and intellectual disability. The proximal muscles of her extremities showed atrophy and mild weakness, and Gowers' sign and gait disturbance were observed. She had no facial muscle involvement or joint contractures. From the age of 2 years and 2 months, she started to experience flaccid paralytic attacks every 4-5 days. The laboratory examination revealed hypokalemia during the attacks. Non-ictal potassium was normal and creatinine kinase was elevated at 200-600 U/L. Genetic analysis revealed a heterozygous variant c.2336G>A (p.S779N) of ATP1A2 , which is the same variant previously reported to cause HPP with intellectual disability. Muscle biopsy showed marked fiber size variation, scattered necrotic and regenerating fibers, increased internal nuclei and vacuoles. Interestingly, tubular aggregate like deposits were seen at the margins of the myofibers. Additionally, electron microscopy revealed severely fragmented nuclei with condensed chromatin, collapsed nuclear membrane, and nucleophagy. This case is the second report of ATP1A2 variant in HPP. The pathological findings, in particular nuclear abnormalities are remarkable, expanding the clinicopathological phenotype associated with ATP1A2 variant. [ABSTRACT FROM AUTHOR]

A neonatal patient with Herpes simplex virus type-2 meningoencephalitis was treated by high-dose intravenous acyclovir therapy. Serum and cerebrospinal fluid (CSF) concentrations were measured retrospectively, showing that the CSF-to-serum concentration ratio was 0.67-0.71, which was higher than the previously reported values in other age groups., (Copyright © 2022 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

This study investigates the optimal meropenem (MEM) dosing regimen for critically ill pediatric patients, for which there is a lack of pharmacokinetic (PK) studies. We conducted a retrospective single-center PK and pharmacodynamic (PD) analysis of 34 pediatric intensive care unit patients who received MEM. Individual PK parameters were determined by a two-compartment analysis. The median (range) age and body weight were 1.4 (0.03 to 14.6) years and 8.9 (2.7 to 40.9) kg, respectively, and eight (23.5%) patients received continuous renal replacement therapy (CRRT), three of whom received extracorporeal membrane oxygenation. Renal function, the systemic inflammatory response syndrome (SIRS) score for the clearance (CL), and the use of CRRT for the central volume of distribution ( V c ) were identified as significant covariates. The mean CL, V c , and peripheral volume of distribution ( V p ) were 0.45 liters/kg/h, 0.49 liters/kg, and 0.34 liters/kg, respectively. The mean population CL of MEM increased by 35% in patients with SIRS and V c increased by 66% in patients on CRRT in the final model. Dosing simulations suggested that the standard dosing regimen provided insufficient PD exposures of a 100% free time above the MIC, and higher doses (40 to 80 mg/kg of body weight/dose every 8 h) with a prolonged 3-h infusion were required to ensure the appropriate PD exposures for patients with SIRS. Our PK model indicated that critically ill pediatric patients are at risk of subtherapeutic exposure under the standard dosing regimen of MEM. A larger, prospective investigation confirming the safety and efficacy of higher concentrations and prolonged infusion of MEM is necessary., (Copyright © 2021 American Society for Microbiology.)

Objectives: Pharmacokinetic (PK) parameters can change significantly during extracorporeal membrane oxygenation (ECMO) and continuous haemodialysis. This case report describes the pharmacokinetics of a 3-h meropenem infusion in an infantile anuric patient on ECMO with continuous haemodialysis., Case: A 19-month-old female patient with asplenia syndrome was admitted to the paediatric intensive care unit for postoperative management of an extracardiac total cavopulmonary connection procedure. Veno-arterial ECMO and continuous haemodialysis were initiated on postoperative Day 2 for circulatory insufficiency due to septic shock and thrombosis of the inferior vena cava extending to the pulmonary artery. Blood and ascites cultures were positive for extended-spectrum β-lactamase-producing Escherichia coli, and 3-h meropenem infusions [120-300 mg/kg/day divided every 8 h (q8h)] were commenced. Following dose escalation to 300 mg/kg/day q8h, sustained negative blood cultures were confirmed. The estimated meropenem clearance and volume of distribution (V d ) were 2.21 mL/kg/min and 0.59 L/kg, respectively. These patient-specific PK parameters were used to predict the PK profile of various dosing regimens. Both 1-h and 3-h infusions of meropenem at 60, 120 and 200 mg/kg/day q8h predicted that the free drug concentration would remain above the minimum inhibitory concentration (fT >MIC ) at an MIC of 1 μg/mL for >40% of the dosing interval. However, when the target was set at 100% fT >MIC , only a 3-h infusion of 200 mg/kg/day q8h could achieve the target in this patient despite the presence of anuria., Conclusion: To optimise meropenem dosing in paediatric patients on ECMO and continuous haemodialysis, further study and PK monitoring are warranted., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

We report on an 8-year-old girl with Wilson disease who developed three episodes of peritonitis due to extended-spectrum beta-lactamase-producing Escherichia coli after liver transplantation. Massive ascites were thought to account for low meropenem concentrations with standard dosing. Extending the infusion achieved higher troughs, greater time above minimum inhibitory concentration., Competing Interests: All authors do not have any potential, perceived, or real conflict of interest., (© 2019 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.)

Understanding anatomical structures and biological functions based on gene expression is critical in a systemic approach to address the complexity of the mammalian brain, where >25 000 genes are expressed in a precise manner. Co-expressed genes are thought to regulate cell type- or region-specific brain functions. Thus, well-designed data acquisition and visualization systems for profiling combinatorial gene expression in relation to anatomical structures are crucial. To this purpose, using our techniques of microtomy-based gene expression measurements and WebGL-based visualization programs, we mapped spatial expression densities of genome-wide transcripts to the 3D coordinates of mouse brains at four post-natal stages, and built a database, ViBrism DB (http://vibrism.neuroinf.jp/). With the DB platform, users can access a total of 172 022 expression maps of transcripts, including coding, non-coding and lncRNAs in the whole context of 3D magnetic resonance (MR) images. Co-expression of transcripts is represented in the image space and in topological network graphs. In situ hybridization images and anatomical area maps are browsable in the same space of 3D expression maps using a new browser-based 2D/3D viewer, BAH viewer. Created images are shareable using URLs, including scene-setting parameters. The DB has multiple links and is expandable by community activity.

Background: Assignment of anatomical reference is a key step in integration of the rapidly expanding collection of rodent brain data. Landmark-based registration facilitates spatial anchoring of diverse types of data not suitable for automated methods operating on voxel-based image information., New Tool: Here we propose a standardized set of anatomical landmarks for registration of whole brain imaging datasets from the mouse and rat brain, and in particular for integration of experimental image data in Waxholm Space (WHS)., Results: Sixteen internal landmarks of the C57BL/6J mouse brain have been reliably identified: by different individuals, independent of their experience in anatomy; across different MRI contrasts (T1, T2, T2(*)) and other modalities (Nissl histology and block-face anatomy); in different specimens; in different slice acquisition angles; and in different image resolutions. We present a registration example between T1-weighted MRI and the mouse WHS template using these landmarks and reaching fairly high accuracy. Landmark positions identified in the mouse WHS template are shared through the Scalable Brain Atlas, accompanied by graphical and textual guidelines for locating each landmark. We identified 14 of the 16 landmarks in the WHS template for the Sprague Dawley rat., Comparison With Existing Methods: This landmark set can withstand substantial differences in acquisition angle, imaging modality, and is less vulnerable to subjectivity., Conclusions: This facilitates registration of multimodal 3D brain data to standard coordinate spaces for mouse and rat brain taking a step toward the creation of a common rodent reference system; raising data sharing to a qualitatively higher level., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Using a recently invented technique for gene expression mapping in the whole-anatomy context, termed transcriptome tomography, we have generated a dataset of 36,000 maps of overall gene expression in the adult-mouse brain. Here, using an informatics approach, we identified a broad co-expression network that follows an inverse power law and is rich in functional interaction and gene-ontology terms. Our framework for the integrated analysis of expression maps and graphs of co-expression networks revealed that groups of combinatorially expressed genes, which regulate cell differentiation during development, were present in the adult brain and each of these groups was associated with a discrete cell types. These groups included non-coding genes of unknown function. We found that these genes specifically linked developmentally conserved groups in the network. A previously unrecognized robust expression pattern covering the whole brain was related to the molecular anatomy of key biological processes occurring in particular areas.

Increased information on the encoded mammalian genome is expected to facilitate an integrated understanding of complex anatomical structure and function based on the knowledge of gene products. Determination of gene expression-anatomy associations is crucial for this understanding. To elicit the association in the three-dimensional (3D) space, we introduce a novel technique for comprehensive mapping of endogenous gene expression into a web-accessible standard space: Transcriptome Tomography. The technique is based on conjugation of sequential tissue-block sectioning, all fractions of which are used for molecular measurements of gene expression densities, and the block- face imaging, which are used for 3D reconstruction of the fractions. To generate a 3D map, tissues are serially sectioned in each of three orthogonal planes and the expression density data are mapped using a tomographic technique. This rapid and unbiased mapping technique using a relatively small number of original data points allows researchers to create their own expression maps in the broad anatomical context of the space. In the first instance we generated a dataset of 36,000 maps, reconstructed from data of 61 fractions measured with microarray, covering the whole mouse brain (ViBrism: http://vibrism.riken.jp/3dviewer/ex/index.html) in one month. After computational estimation of the mapping accuracy we validated the dataset against existing data with respect to the expression location and density. To demonstrate the relevance of the framework, we showed disease related expression of Huntington's disease gene and Bdnf. Our tomographic approach is applicable to analysis of any biological molecules derived from frozen tissues, organs and whole embryos, and the maps are spatially isotropic and well suited to the analysis in the standard space (e.g. Waxholm Space for brain-atlas databases). This will facilitate research creating and using open-standards for a molecular-based understanding of complex structures; and will contribute to new insights into a broad range of biological and medical questions.

Background: Mesothelioma is a highly malignant tumor that is primarily caused by occupational or environmental exposure to asbestos fibers. Despite worldwide restrictions on asbestos usage, further cases are expected as diagnosis is typically 20-40 years after exposure. Once diagnosed there is a very poor prognosis with a median survival rate of 9 months. Considering this the development of early pre clinical diagnostic markers may help improve clinical outcomes., Methodology: Microarray expression arrays on mesothelium and other tissues dissected from mice were used to identify candidate mesothelial lineage markers. Candidates were further tested by qRTPCR and in-situ hybridization across a mouse tissue panel. Two candidate biomarkers with the potential for secretion, uroplakin 3B (UPK3B), and leucine rich repeat neuronal 4 (LRRN4) and one commercialized mesothelioma marker, mesothelin (MSLN) were then chosen for validation across a panel of normal human primary cells, 16 established mesothelioma cell lines, 10 lung cancer lines, and a further set of 8 unrelated cancer cell lines., Conclusions: Within the primary cell panel, LRRN4 was only detected in primary mesothelial cells, but MSLN and UPK3B were also detected in other cell types. MSLN was detected in bronchial epithelial cells and alveolar epithelial cells and UPK3B was detected in retinal pigment epithelial cells and urothelial cells. Testing the cell line panel, MSLN was detected in 15 of the 16 mesothelioma cells lines, whereas LRRN4 was only detected in 8 and UPK3B in 6. Interestingly MSLN levels appear to be upregulated in the mesothelioma lines compared to the primary mesothelial cells, while LRRN4 and UPK3B, are either lost or down-regulated. Despite the higher fraction of mesothelioma lines positive for MSLN, it was also detected at high levels in 2 lung cancer lines and 3 other unrelated cancer lines derived from papillotubular adenocarcinoma, signet ring carcinoma and transitional cell carcinoma.

Precise patterning of morphogen molecules and their accurate reading out are of key importance in embryonic development. Recent experiments have visualized distributions of proteins in developing embryos and shown that the gradient of concentration of Bicoid morphogen in Drosophila embryos is established rapidly after fertilization and remains stable through syncytial mitoses. This stable Bicoid gradient is read out in a precise way to distribute Hunchback with small fluctuations in each embryo and in a reproducible way, with small embryo-to-embryo fluctuation. The mechanisms of such stable, precise, and reproducible patterning through noisy cellular processes, however, still remain mysterious. To address these issues, here we develop the one- and three-dimensional stochastic models of the early Drosophila embryo. The simulated results show that the fluctuation in expression of the hunchback gene is dominated by the random arrival of Bicoid at the hunchback enhancer. Slow diffusion of Hunchback protein, however, averages out this intense fluctuation, leading to the precise patterning of distribution of Hunchback without loss of sharpness of the boundary of its distribution. The coordinated rates of diffusion and transport of input Bicoid and output Hunchback play decisive roles in suppressing fluctuations arising from the dynamical structure change in embryos and those arising from the random diffusion of molecules, and give rise to the stable, precise, and reproducible patterning of Bicoid and Hunchback distributions.

Inactivation of serotonin transporter (HTT) by pharmacologically in the neonate or genetically increases risk for depression in adulthood, whereas pharmacological inhibition of HTT ameliorates symptoms in depressed patients. The differing role of HTT function during early development and in adult brain plasticity in causing or reversing depression remains an unexplained paradox. To address this we profiled the gene expression of adult Htt knockout (Htt KO) mice and HTT inhibitor-treated mice. Inverted profile changes between the two experimental conditions were seen in 30 genes. Consistent results of the upstream regulatory element search and the co-localization search of these genes indicated that the regulation may be executed by Pax5, Pax7 and Gata3, known to be involved in the survival, proliferation, and migration of serotonergic neurons in the developing brain, and these factors are supposed to keep functioning to regulate downstream genes related to serotonin system in the adult brain.

Background: Recent analyses have suggested that many genes possess multiple transcription start sites (TSSs) that are differentially utilized in different tissues and cell lines. We have identified a huge number of TSSs mapped onto the mouse genome using the cap analysis of gene expression (CAGE) method. The standard hierarchical clustering algorithm, which gives us easily understandable graphical tree images, has difficulties in processing such huge amounts of TSS data and a better method to calculate and display the results is needed., Results: We use a combination of hierarchical and non-hierarchical clustering to cluster expression profiles of TSSs based on a large amount of CAGE data to profit from the best of both methods. We processed the genome-wide expression data, including 159,075 TSSs derived from 127 RNA samples of various organs of mouse, and succeeded in categorizing them into 70-100 clusters. The clusters exhibited intriguing biological features: a cluster supergroup with a ubiquitous expression profile, tissue-specific patterns, a distinct distribution of non-coding RNA and functional TSS groups., Conclusion: Our approach succeeded in greatly reducing the calculation cost, and is an appropriate solution for analyzing large-scale TSS usage data.

To thoroughly understand the function and regulation of neurotransmitter systems in the brain, as well as the underlying disease mechanisms, it is important to comprehensively analyze the expression patterns of genes participating in such systems. Using functional annotated cDNA clones (FANTOM), we examined the gene expression patterns of the serotonin neurotransmitter system, which is involved in psychiatric diseases such as depression. We chose 24 gene products and visualized their endogenous localizations using in situ hybridization (ISH). We were able to fine-tune an automated ISH method to obtain high-resolution cell-based figures within 24 h. We also measured the amounts of mRNAs with quantitative RT-PCR. The outline of the in situ gene expression pattern viewed under low magnification agreed with the results of the RT-PCR. In the high-resolution view obtained with ISH, we could document novel localizations of the several genes critically related to serotonin activity.

Background: An antisense transcript of histone H2a that has no significant protein-coding region has been cloned from a mouse full-length cDNA library. In the present study, we evaluated this transcript by using RT-PCR and compared the expression patterns of the sense and antisense transcripts by using quantitative RT-PCR (qRT-PCR)., Results: This antisense RNA was expressed in three mouse cell lines. We call it ASH2a. ASH2a includes not only the complementary sequence of the transcript of Hist2h2aa2 (a replication-dependent histone H2a gene), but also that of the promoter of Hist2h2aa2. The upstream genomic sequence of the transcription start site of the ASH2a-coding gene (ASH2a) lacks both CCAAT and TATA boxes. This absence suggests that the regulation of ASH2a is different from that of the replication-dependent histone H2a genes. Findings from qRT-PCR indicated that the expression pattern of ASH2a was different from that of Hist2h2aa2. Expression of Hist2h2aa2 peaked at 2 to 4 h during S-phase, but that of ASH2a peaked at 1 h., Conclusion: We showed the existence of ASH2a, a histone H2a antisense RNA, in mouse cultured cells. The expression pattern of ASH2a is different from that of the sense RNA.

Insulin receptor tyrosine kinase substrate of 53-kDa protein (IRSp53) is now known to be a key factor in cytoskeleton reorganization. The human IRSp53 was identified as a binding partner with DRPLA protein, a product of the gene responsible for a neurodegenerative disorder, dentatorubral pallidoluysian atrophy, as well as a binding partner with brain-specific angiogenesis inhibitor 1. Previous studies identified at least four isoforms (L-, M-, S- and T-forms) in human, where 511 amino acid residues from the N-terminus were identical, followed by unique sequences of 9-41 amino acid residues. As each isoform had a distinct function, the unique sequences at the C-terminus had a vital role in its function. Here we report that these isoforms were indeed generated by alternative splicing, which was established by experimental and computational studies on human and rodent genomes. Previous biochemical reports suggested that rodents may lack one of the isoforms (L-form). This study solved this issue, as a nucleotide substitution occurred at a splice donor site followed by a large deletion in the rodent genome compared with human, which made the generation of the L-form impossible. This study also revealed overlapping of the IRSp53 and AATK genes coded for by complementary strands.

An insulin-receptor substrate of 53-kDa protein (IRSp53) is an adapter protein, which interacts with the Rho-family of GTPases and mediates neurite outgrowth. It also binds to DRPLA protein, a product of the gene responsible for a polyglutamine disease, dentatorubral-pallidoluysian atrophy (DRPLA). Isoforms of human IRSp53 have been reported, each with a unique amino acid sequence at the C-terminal end. Here we report the distinctive tissue distribution and phosphorylation of three isoforms (L, S, and T-forms). Western blotting analyses with isoform-specific antibodies demonstrated that the L and S-forms were expressed in the brain, whereas the T-form was not present in any tissues examined, but was found in a cancer cell line. The L and S-forms were phosphorylated upon stimulation with insulin, and the T-form with IGF-I. Since phospho-acceptor sites were localized to the common portion, the difference in phosphorylation seems to be due to the unique C-terminal sequence.

We have identified and characterized three missense mutations in a patient with type 1 G(M1) gangliosidosis, namely a substitution of G for A at nucleotide position 1044 (G1044-->A; in exon 10) on one allele, which converts Asp(332) into asparagine, and both a mutation (C492-->A in exon 4, leading to the amino acid change of Arg(148)-->Ser) and a polymorphism (A1644-->G in exon 15, leading to a change of Ser(532)-->Gly) on the other allele. This patient had less than 1% residual beta-galactosidase activity and minimally detectable levels of immunoreactive beta-galactosidase protein in fibroblasts. To account for the above findings, a series of expression and immunolocalization studies were undertaken to assess the impact of each mutation. Transient overexpression in COS-1 cells of cDNAs encoding Asp(332)Asn, Arg(148)Ser and Ser(532)Gly mutant beta-galactosidases produced abundant amounts of precursor beta-galactosidase, with activities of 0, 84 and 81% compared with the cDNA clone for wild-type beta-galactosidase (GP8). Since the level of vector-driven expression is much less in Chinese hamster ovary (CHO) cells than in COS-1 cells, and we knew that exogenous beta-galactosidase undergoes lysosomal processing when expressed in these cells, transient expression studies were performed of Arg(148)Ser and Ser(532)Gly, which yielded active forms of the enzyme. In this case, the Arg(148)Ser and Ser(532)Gly products gave rise to 11% and 86% of the control activity respectively. These results were not unexpected, since the Arg(148)Ser mutation introduced a major conformational change into the protein, and we anticipated that it would be degraded in the endoplasmic reticulum (ER), whereas the polymorphism was expected to produce near-normal activity. To examine the effect of the Asp(332)Asn mutation on the catalytic activity, we isolated CHO clones permanently transfected with the Asp(332)Asn and Asp(332)Glu constructs, purified the enzymes by substrate-analogue-affinity chromatography, and determined their kinetic parameters. The V(max) values of both mutant recombinant enzymes were markedly reduced (less than 0.9% of the control), and the K(m) values were unchanged compared with the corresponding wild-type enzyme isolated at the same time. Both the Arg(148)Ser beta-galactosidase in CHO cells and Asp(332)Asn beta-galactosidases (in COS-1 and CHO cells) produced abundant immunoreaction in the perinuclear area, consistent with localization in the ER. A low amount was detected in lysosomes. Incubation of patient fibroblasts in the presence of leupeptin, which reduces the rate of degradation of lysosomal beta-galactosidase by thiol proteases, had no effect on residual enzyme activity, and immunostaining was again detected largely in the perinuclear area (localized to the ER) with much lower amounts in the lysosomes. In summary, the Arg(148)Ser mutation has no effect on catalytic activity, whereas the Asp(332)Asn mutation seriously reduces catalytic activity, suggesting that Asp(332) might play a role in the active site. Immunofluorescence studies indicate the expressed mutant proteins with Arg(148)Ser and Asp(332)Asn mutations are held up in the ER, where they are probably degraded, resulting in only minimum amounts of the enzyme becoming localized in the lysosomes. These results are completely consistent with findings in the cultured fibroblasts. Our results imply that most of the missense mutations described in G(M1) gangliosidosis to date have little effect on catalytic activity, but do affect protein conformation such that the resulting protein cannot be transported out of the ER and fails to arrive in the lysosome. This accounts for the minimal amounts of enzyme protein and activity seen in most G(M1) gangliosidosis patient fibroblasts.

Dentatorubral-pallidoluysian atrophy (DRPLA) is one of the hereditary neurodegenerative disorders caused by expansion of CAG/glutamine repeats. To investigate the normal function of the DRPLA gene and the pathogenic mechanism of neuron death in specific areas of the brain, we isolated and analyzed a gene that shares a notable motif with DRPLA, arginine-glutamic acid (RE) dipeptide repeats. The gene isolated, designated RERE, has an open reading frame of 1566 amino acids, of which the C-terminal portion has 67% homology to DRPLA, whereas the N-terminal portion is distinctive. RERE also contains arginine-aspartic acid (RD) dipeptide repeats and putative nuclear localization signal sequences, but no polyglutamine tracts. RERE is expressed at a low level in most tissues examined. Immunoprecipitation and in vitro binding assays demonstrate that the DRPLA and RERE proteins bind each other, for which one of the RE repeats has a primary role, and extended polyglutamine enhances the binding. With engineered constructs fused with a tag, the RERE protein localized predominantly in the nucleus. Moreover, when RERE is overexpressed, the distribution of endogenous DRPLA protein alters from the diffused to the speckled pattern in the nucleus so as to co-localize with RERE. More RERE protein is recruited into nuclear aggregates of the DRPLA protein with extended polyglutamine than into those of pure polyglutamine. These results reveal a function for the DRPLA protein in the nucleus and the RE repeat in the protein-protein interaction.

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases., (Copyright 1999 Academic Press.)

HTLV-I infection is a recently recognized disease entity that is common in some tropical and subtropical areas, including the southwestern district of Japan. Despite the geographical proximity and frequent cultural exchanges between Korea and Japan, it is understood that Korea is not an endemic area and HTLV-I-associated illnesses are very rare in Korea. This study was designed to evaluate the positive rate of anti-HTLV-I antibodies in Korean blood donors and its regional distribution. Sera were obtained from blood donors from various districts around Korea. Anti-HTLV-I antibodies were detected by using the microtiter particle agglutination test employing an indirect agglutination technique. A total of 9,281 donors were tested and 12 donors (0.13%) were positive for anti-HTLV-I antibodies, 10 (0.11%) out of 8,845 males and 2 (0.46%) out of 436 females, with relative female predominance. A relatively high incidence of anti-HTLV-I positive donors was observed in Cheju Island (0.80%), Kyungnam (0.31%), and Chonnam (0.15%). In conclusion, the positive rate of anti-HTLV-I antibodies seemed to be very low in Korea, but the highest positive rate of anti-HTLV-I antibodies was noticed on Cheju Island, warranting further research for confirmation.

Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder caused by the abnormal CAG triplet-repeat expansion resulting in an elongated polyglutamine (polyQ) stretch. We have recently showed that the DRPLA protein is cleaved during apoptosis by caspase-3, one of the cysteine protease family members known to be activated during apoptosis. We report here the subcellular localization of the DRPLA protein by fusing the green fluorescent protein as a tag. The full length DRPLA protein is localized predominantly but not exclusively in the nucleus regardless of the length of the polyQ stretch. In contrast, an N-terminal-deleted fragment containing polyQ produced by the proteolytic cleavage with caspase-3 is found both in the nucleus and the cytoplasm. Moreover, the same fragment with the elongated polyQ showed aggregation when overexpressed. Some cells with aggregate formation showed apoptotic phenotype. These findings raise the possibility that the DRPLA protein processed by caspase-3 may lead to aggregation of the protein resulting in the development of neurodegeneration.

Lysosomal beta-galactosidase precursor is processed to a mature form and associated with protective protein in lysosomes. In this study we used two cysteine protease proinhibitors, E64-d for cathepsins B, S, H, and L, and CA074Me for cathepsin B. They are converted intracellularly to active forms, E-64c and CA074, respectively. Both active compounds inhibited maturation of the exogenous beta-galactosidase precursor, but E-64c did not inhibit further degradation to an inactive 50-kDa product. We concluded that cathepsin B participated exclusively in maturation of beta-galactosidase, and a non-cysteine protease was involved in further degradation and inactivation of the enzyme molecule.

Processing of human beta-galactosidase (beta-GAL) was studied in permanently transfected Chinese hamster ovary (CHO) cells and compared with that in normal cells and in cells from subjects with GM1-gangliosidosis, galactosialidosis and I-cell disease. Biosynthesis of beta-GAL in CHO cells results in the synthesis of an 88 kDa glycosylated and phosphorylated monomer precursor which is enzymically active and is secreted into the medium. Post-translational processing begins at the C-terminal end of the protein and gives rise to structurally related 67 and 64 kDa mature forms. These are subsequently degraded to give several inactive products of which a 50 kDa and a 18 kDa species are prominent. In normal fibroblasts only the 84 kDa precursor is readily detected inside cells, while the 88 kDa precursor is the only form secreted from cells in the presence of ammonium chloride. Processing of the precursor in normal fibroblasts results in the appearance of both the 67 and 64 kDa mature forms, which are also degraded to give 50 and 18 kDa products, as in transfected CHO cells. As affected controls, GM1-gangliosidosis cells showed a general loss of all forms of the enzyme, while in I-cell fibroblasts only the 84 kDa precursor and an 18 kDa degradation form were prominent. In galactosialidosis fibroblasts, taken from two different subjects, processing of beta-GAL was characterized by the respective appearance of intermediate 80 and 72 kDa enzymically inactive polypeptides, at levels lower than the normal amounts of the 67 and 64 kDa mature forms and higher than the normal amounts of degradation products, one of which is of 45 kDa and arises by endoproteolytic cleavage of the 80 kDa polypeptide. Incubation for up to 72 h in medium containing leupeptin, a potent inhibitor of thiol-dependent proteases, resulted in a significantly increased level of beta-GAL activity to near normal levels in fibroblasts from one galactosialidosis subject. Concordant with this, the abundance of the 84 kDa precursor was increased and the levels of the 80 kDa, 45 kDa and 18 kDa digestion products were diminished. However, in fibroblasts from the second galactosialidosis subject, the amount of the abnormal 72 kDa polypeptide was not influenced by leupeptin treatment. Leupeptin treatment did not increase enzymic activity levels in normal, GM1-gangliosidosis or I-cell disease fibroblasts, despite the fact that the production of the 50 kDa and 18 kDa degradation products was blocked in the presence of leupeptin. We concluded that in galactosialidosis the leupeptin-inhibitable proteolytic cleavage of a small fragment causes a conformational change of the precursor that precludes its further normal processing and results in its enzymic deficiency. This early abnormal trimming of beta-GAL is ascribable to a deficiency in the functional protective protein, the function of which is absolutely essential to render beta-GAL cryptic from at least two distinct and separate proteolytic attacks that together remove at least 12 kDa from the C-terminal end of the enzyme.

Chinese hamster ovary cell clones permanently transfected with the cDNA for human lysosomal beta-galactosidase secrete the enzyme precursor into the cell medium, from which it is purified to apparent homogeneity in a single step by affinity chromatography. The purified precursor is fully active, displays the same pH optimum and Km values as the mature placental enzyme, and has an intact C-terminus. The intact enzyme when chromatographed on a Sephacryl S-200 molecular-sieve column elutes as a 105,500 Da monomer, whereas on SDS/PAGE gels the polypeptide migrates as an 88 kDa polypeptide. A time course of digestion with glycopeptide-N-glycanase shows the gradual conversion of the precursor from an 88 to a 72 kDa protein, suggesting the presence of five N-linked oligosaccharides in the protein. The precursor is readily taken up in a mannose-6-phosphate-dependent manner into beta-galactosidase-deficient, GM1-gangliosidosis fibroblasts, and the enzyme activity is returned to normal levels. We show that the stereochemical course of enzymic hydrolysis involves the retention of the beta-configuration at the anomeric centre, suggesting a double-displacement mechanism. Furthermore, the enzyme is rapidly and irreversibly inactivated in the presence of the mechanism-based inactivator 2,4-dinitrophenyl-2-deoxy-2-fluoro-beta-D-galactopyranoside, which implicates a covalent intermediate. The enzyme is also inactivated by 1-ethyl-3(3-dimethylamino-propyl)carbodi-imide and by phenylglyoxal, which implicates carboxylate and arginine residues respectively in the active site. We conclude that the beta-galactosidase precursor is functionally identical to the mature lysosomal form of the enzyme and serves as an excellent enzyme source for investigation of structure-function relationships in the protein.