Chemical chaperone therapy: chaperone effect on mutant enzyme and cellular pathophysiology in ß-galactosidase deficiency.

ß-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G(M1) -gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid ß-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant ß-galactosidase proteins. In this study, we performed genotype analyses of patients with ß-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant ß-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify ß-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human ß-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G(M1) accumulation and ameliorated the impairments of lipid trafficking and protein degradation in ß-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for ß-galactosidase deficiency.

Possible use of RNA isolate from inactivated vaccine for external positive control in reverse transcription-based detection of foot-and-mouth disease virus in bull semen.

This study has demonstrated the novel use of inactivated and purified vaccine against FMD virus for detection and analysis. RNA isolate was efficiently generated from the vaccine for an external positive control for reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays. The target DNA fragment sequences from the 2B region and 3D RNA polymerase gene of the virus for RT-PCR and RT-LAMP respectively were successfully amplified using the RNA template. Laboratories lacking complex equipment may not be feasible to handle high-risk viruses for conventional methods such as the isolation and culture of live viruses. Here, with the use of these molecular tools, novel use of RNA isolate from inactivated, purified vaccine proved to be an effective external positive control for the assays. Therefore, with these methods, the derived RNA control template aids in a safe method for screening FMD virus for diagnostic laboratories. And by using the same technique, it is then possible to generate a standard for diagnosing any other infectious viral diseases.

Enhanced autophagic cell death in expanded polyhistidine variants of HOXA1 reduces PBX1-coupled transcriptional activity and inhibits neuronal differentiation.

HOXA1 is a member of the homeobox gene family and is involved in early brain development. In our previous study, we identified novel variants of polyhistidine repeat tract in HOXA1 gene and showed that ectopic expression of expanded variants led to enhanced intranuclear aggregation and accelerated cell death in a time-dependent manner. Here, we further investigate the implications of polyhistidine variants on HOXA1 function. Aside from intranuclear aggregation, we observed cytosolic aggregates during the early stages of expression. Rapamycin, an autophagy inducer, resulted in decreased protein aggregation and cell death. Here, we also show an interaction between variants of HOXA1 and one of the HOX protein known cofactors, PBX1. Expanded HOXA1 variants exhibited reduced PBX1-coupled transcriptional activity through a regulatory enhancer of HOXB1. Moreover, we demonstrate that both deleted and expanded variants inhibited neurite outgrowth in retinoic acid-induced neuronal differentiation in neuroblastoma cells. These results provide further evidence that expanded polyhistidine repeats in HOXA1 enhance aggregation and cell death, resulting in impaired neuronal differentiation and cooperative binding with PBX1.

Polyhistidine tract expansions in HOXA1 result in intranuclear aggregation and increased cell death.

HOXA1 gene is part of a cluster of homeotic selector genes that regulates the anteroposterior patterning of mammals during embryonic development. HOXA1 encodes two alternatively spliced mRNAs with two isoforms, A and B, the former contains the homeodomain and expressed in early embryonic development. HOXA1 contains a string of 10 histidine repeats. However, individuals heterozygous for 7, 9, 11, and 12 histidine repeat variants were present among the Japanese population, notably in some autism cases. To determine the biological implications of the different polyhistidine repeat lengths, we expressed these variants in COS-7 and a human neuroblastoma cell line (SK-N-SH). Expression of expanded variants of HOXA1 isoform A, containing 11 and 12 polyhistidine, resulted in early and great degree of protein aggregation in the nucleus. This aggregation resulted in accelerated cell death in cells expressing 11 and 12 expanded variants compared to those transfected with 7 and 10 polyhistidine variants. Furthermore, we showed that these aggregates were ubiquitinated and were inhibited by a histidine-modifying compound, DEPC. These data suggest that HOXA1 protein with polyhistidine tract expansions misfold, aggregate, and have a toxic effect on cell.

Genetic diversity of JC virus in the modern Filipino population: implications for the peopling of the Philippines.

The Philippines is generally believed to have been established by various peoples who migrated from neighboring areas. To gain new insights into the peopling of the Philippines, we used the JC virus (JCV) genotyping approach. We collected about 50 urine samples on each of two representative islands of the Philippines, Luzon and Cebu. DNA was extracted from the urine samples and used to amplify the 610-bp region (IG region) of the viral genome. For each island, we determined about 20 IG sequences, from which a neighbor-joining phylogenetic tree was constructed to classify the JCV isolates detected into distinct genotypes. The predominant genotype detected was SC, the Southeast Asian genotype. Minor JCV genotypes were SC/Phi, B1-a, and B3. SC/Phi was a subcluster of SC and has not been detected in areas other than the Philippines. B1-a was detected previously in mainland China, Pamalican Island (Palawan, Philippines), and Taiwan (an aboriginal tribe). B3 was classified in this study into two subgroups, one (B3-a) containing three Luzon isolates and several Chinese, Thai, and Uzbek isolates, the other (B3-b) containing two Luzon, one Cebu, and one Indonesian isolate. These findings suggest that the modern Filipino population was formed not only by Southeast Asians carrying SC but also by a few distinct ethnic groups carrying SC/Phi, B1-a, and B3-a or -b.