Roles of Fukutin, the Gene Responsible for Fukuyama-Type Congenital Muscular Dystrophy, in Neurons: Possible Involvement in Synaptic Function and Neuronal Migration

Fukutin is a gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), accompanying ocular and brain malformations represented by cobblestone lissencephaly. Fukutin is related to basement membrane formation via the glycosylation of α-dystoglycan (α-DG), and astrocytes play a crucial role in the pathogenesis of the brain lesion. On the other hand, its precise function in neurons is unknown. In this experiment, the roles of fukutin in mature and immature neurons were examined using brains from control subjects and FCMD patients and cultured neuronal cell lines. In quantitative PCR, the expression level of fukutin looked different depending on the region of the brain examined. A similar tendency in DG expression appears to indicate a relation between fukutin and α-DG in mature neurons. An increase of DG mRNA and core α-DG in the FCMD cerebrum also supports the relation. In immunohistochemistry, dot-like positive reactions for VIA4-1, one of the antibodies detecting the glycosylated α-DG, in Purkinje cells suggest that fukutin is related to at least a post-synaptic function via the glycosylation of α-DG. As for immature neurons, VIA4-1 was predominantly positive in cells before and during migration with expression of fukutin, which suggest a participation of fukutin in neuronal migration via the glycosylation of α-DG. Moreover, fukutin may prevent neuronal differentiation, because its expression was significantly lower in the adult cerebrum and in differentiated cultured cells. A knockdown of fukutin was considered to induce differentiation in cultured cells. Fukutin seems to be necessary to keep migrating neurons immature during migration, and also to support migration via α-DG

Ex Vivo Expansion of Human Hematopoietic Stem Cells by Garcinol, a Potent Inhibitor of Histone Acetyltransferase

BACKGROUND: Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34(+)CD38(-) HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34(+)CD38(-) HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34(+) HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not. CONCLUSIONS/SIGNIFICANCE: Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs

Antisense PMO Found in Dystrophic Dog Model Was Effective in Cells from Exon 7-Deleted DMD Patient

BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD). We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO) targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J)) lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J) and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J) and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans

Effect of sitting position on respiratory status in preterm infants

Objective: To evaluate whether using a sitting-type car safety seats for preterm infants is advisable. Patients and methods: A total of 65 preterm infants underwent polysomnography in the supine and sitting positions. The infants with <95% of SpO2 were assigned to the desaturation (DS) group. Their backgrounds, breathing patterns, and breathing types were analyzed. Results: Of the 65 cases, 18 were assigned to the DS group. No significant differences were observed between the DS and non-DS groups in their background characteristics. Of the 18 DS cases, 15 were included in the non-obstructive group (8, periodic breathing; 6, tachypnea; 1, irregular breathing). Conclusions: Immaturity of the respiratory center and decompensation for the respiratory changes caused by the sitting position were suspected to cause DS in infants. Sitting-type car safety seats should be used with caution for preterm infants, and all preterm infants need to be screened by polysomnographic examination in the sitting position.Peer Reviewe

Fabry Disease: Novel α-Galactosidase A 3′-Terminal Mutations Result in Multiple Transcripts Due to Aberrant 3′-End Formation

Mutations in the gene that encodes the lysosomal exoglycohydrolase, α-galactosidase A (α-GalA), cause Fabry disease, an X-linked recessive inborn error of glycosphingolipid catabolism. Human α-GalA is one of the rare mammalian genes that has its polyadenylation signal in the coding sequence and lacks a 3′ untranslated region (UTR). We identified two novel frameshift mutations, 1277delAA (del2) and 1284delACTT (del4), in unrelated men with classical Fabry disease. Both mutations occurred in the 3′ terminus of the coding region and obliterated the termination codon, and del2 also altered the polyadenylation signal. To characterize these mutations, 3′ rapid amplification of cDNA ends (RACE) and polymerase chain reactions (PCR) were performed, and the amplicons were subcloned and sequenced. Both mutations generated multiple transcripts with various lengths of 3′ terminal sequences, some elongating ∼1 kb. Mutant transcripts were classified as follows: type I transcripts had terminal in-frame thymidines that created termination codons when polyadenylated, type II had downstream termination codons within the elongated α-GalA sequence, and type III, the most abundant, lacked termination codons at their 3′ ends. To determine if the type III transcripts were degraded by the recently described cytosolic messenger RNA degradation pathway for messages lacking termination codons, northern blot analysis was performed. However, the finding of similar levels of nuclear and cytoplasmic α-GalA mRNA in normal and patient lymphoblasts suggested that mRNA degradation did not result from either mutation. Expression of representative transcript types revealed differences in intracellular localization and/or protein stability and catalytic activity, with most mutant proteins being nonfunctional. Characterization of these 3′ mutations identified a novel molecular mechanism causing classical Fabry disease

てんかん性無呼吸発作を認めたBrachmann-de Lange症候群の1乳児例(<特集>小児科学教室大澤真木子教授開講5周年記念)

乳児期の無呼吸発作には様々な原因があるが,てんかん性無呼吸発作は極めて稀である.一方,Brachmann-de Lange症候群の約20%にてんかん発作を認めるが,我々の調べた限りではてんかん性無呼吸発作を伴った症例は報告されていない.我々は,頻回のてんかん性無呼吸発作を認めたBrachmann-de Lange症候群の1乳児例の発作時ポリグラフについて報告する.発作時ポリグラフでは,無呼吸に先だって左前頭極部に突発性θ波が認められた.部分発作は,無呼吸発作の鑑別疾患の一つであり,発作時ポリグラフが診断の唯一の方法である.We describe a case of Brachmann-de Lange syndrome in an infant who had repeated life-threatening episodes of epileptic apnea, and we report a polygraphic recording made while monitoring changes in oxygen saturation. Focal ictal paroxysmal theta-wave activity in the left frontal pole region preceded the apnea. Partial seizures should be included in the differential diagnosis of apnea, and the only way to make a conclusive diagnosis is by polygraphic ictal EEG recording during an apneas spell.小児科学教室大澤真木子教授開講5周年記念特

てんかん性無呼吸発作を認めたBrachmann-de Lange症候群の1乳児例(<特集>小児科学教室大澤真木子教授開講5周年記念)

乳児期の無呼吸発作には様々な原因があるが,てんかん性無呼吸発作は極めて稀である.一方,Brachmann-de Lange症候群の約20%にてんかん発作を認めるが,我々の調べた限りではてんかん性無呼吸発作を伴った症例は報告されていない.我々は,頻回のてんかん性無呼吸発作を認めたBrachmann-de Lange症候群の1乳児例の発作時ポリグラフについて報告する.発作時ポリグラフでは,無呼吸に先だって左前頭極部に突発性θ波が認められた.部分発作は,無呼吸発作の鑑別疾患の一つであり,発作時ポリグラフが診断の唯一の方法である.We describe a case of Brachmann-de Lange syndrome in an infant who had repeated life-threatening episodes of epileptic apnea, and we report a polygraphic recording made while monitoring changes in oxygen saturation. Focal ictal paroxysmal theta-wave activity in the left frontal pole region preceded the apnea. Partial seizures should be included in the differential diagnosis of apnea, and the only way to make a conclusive diagnosis is by polygraphic ictal EEG recording during an apneas spell