Novel autophagy modulators: Design and synthesis of (+)-epogymnolactam analogues and structure-activity relationship

(+)-Epogymnolactam (1) was discovered as a novel autophagy inducer from a culture of Gymnopus sp. in our laboratory. To determine structure-activity relationships among (+)-epogymnolactam analogues comparing with cerulenin (2), we synthesized 5 analogues including (?)-epogymnolactam (3) having each different functional group, and 3 analogues with different side-chain lengths. Five analogues, 3, 4, 5, 6, and 7 did not significantly increase the ratio of LC3-II to LC3-I as an autophagy marker in NIH3T3 cells. These results suggest that presence and stereochemistry of (2R,3S)-epoxy group and cyclic syn-form (1b) of 1 are important for the activity as autophagy inducer. Hexyl analogue (8) as well as 1 having butyl side-chain dose-dependently increased the ratio of LC3-II to LC3-I, whereas octyl analogue (9) and 2 rather decreased the ratio. Decyl analogue (10) did not give a change in the ratio. Although 8 seemed to be an excellent autophagy inducer, it dose-dependently increased SQSTM1 (p62) as in the case of 2, whereas 1 showed a slight dose-dependent decrease of p62 as an index of autophagic protein degradation. These observations suggest that 8 is an autophagy modulator with different molecular target from 1 or 2.     

Conversion of iodine to fluorine-18 based on iodinated chalcone and evaluation for β-amyloid PET imaging

In the amyloid cascade hypothesis, β-amyloid (Aβ) plaques is one of the major pathological biomarkers in the Alzheimer’s disease (AD) brain. We report the synthesis and evaluation of novel radiofluorinated chalcones, [¹⁸F]4-dimethylamino-4′-fluoro-chalcone ([¹⁸F]DMFC) and [¹⁸F]4′-fluoro-4-methylamino-chalcone ([¹⁸F]FMC), as Aβ imaging probes. The conversion of iodine directly introduced to the chalcone backbone into fluorine was successfully carried out by ¹⁸F-labeling via the corresponding boronate precursors, achieving the direct introduction of fluorine-18 into the chalcone backbone to prepare [¹⁸F]DMFC and [¹⁸F]FMC. In a biodistribution study using normal mice, [¹⁸F]DMFC and [¹⁸F]FMC showed a higher initial uptake (4.43 and 5.47% ID/g at 2?min postinjection, respectively) into and more rapid clearance (0.52 and 0.66% ID/g at 30?min postinjection, respectively) from the brain than a Food and Drug Administration (FDA)-approved Aβ imaging agent ([¹⁸F]Florbetapir), meaning the improvement of the probability of detecting Aβ plaques and the reduction of non-specific binding in the brain. In the in vitro binding studies using aggregates of recombinant Aβ peptides, [¹⁸F]DMFC and [¹⁸F]FMC showed high binding affinity to recombinant Aβ aggregates at the K_d values of 4.47 and 6.50?nM, respectively. In the in vitro autoradiography (ARG) experiment with AD brain sections, [¹⁸F]DMFC and [¹⁸F]FMC markedly accumulated only in a region with abundant Aβ plaques, indicating that they clearly recognized human Aβ plaques in vitro. These encouraging results suggest that [¹⁸F]DMFC and [¹⁸F]FMC may be promising PET probes for the detection of an amyloid pathology and the early diagnosis of AD with marked accuracy.     

Comparison of Two Pearl Sacs Formed in the Same Recipient Oyster with Different Genetic Background Involved in Yellow Pigmentation in <Emphasis Type="Italic">Pinctada fucata</Emphasis>

Color is one of the most important factors determining the commercial value of pearls. Pinctada fucata is a well-known pearl oyster producing high-quality Akoya pearls. Phenotypic variation in amount of yellow pigmentation produces white and yellowish pearls. It has been reported that polymorphism of yellow pigmentation of Akoya pearls is genetically regulated, but the responsible gene(s) has remained unknown. Here, we prepared pearl sac pairs formed in the same recipient oyster but coming from donor oysters that differ in their color. These two pearl sacs produced pearls with different yellowness even in the same recipient oyster. Yellow tone of produced pearls was consistent with shell nacre color of donor oysters from which mantle grafts were prepared, indicating that donor oysters strongly contribute to the yellow coloration of Akoya pearls. We also conducted comparative RNA-seq analysis and retrieved several candidate genes involved in the pearl coloration. Whole gene expression patterns of pair sacs were not grouped by pearl color they produced, but grouped by recipient oysters in which they were grown, suggesting that the number of genes involved in the yellow coloration is quite small, and that recipient oyster affects gene expression of the majority of genes in the pearl sac.     

Bone morphogenetic protein signaling through ACVR1 and BMPR1A negatively regulates bone mass along with alterations in bone composition

Bone quantity and bone quality are important factors in determining the properties and the mechanical functions of bone. This study examined the effects of disrupting bone morphogenetic protein (BMP) signaling through BMP receptors on bone quantity and bone quality. More specifically, we disrupted two BMP receptors, Acvr1 and Bmpr1a, respectively, in Osterix-expressing osteogenic progenitor cells in mice. We examined the structural changes to the femora from 3-month old male and female conditional knockout (cKO) mice using micro-computed tomography (micro-CT) and histology, as well as compositional changes to both cortical and trabecular compartments of bone using Raman spectroscopy. We found that the deletion of Acvr1 and Bmpr1a, respectively, in an osteoblast-specific manner resulted in higher bone mass in the trabecular compartment. Disruption of Bmpr1a resulted in a more significantly increased bone mass in the trabecular compartment. We also found that these cKO mice showed lower mineral-to-matrix ratio, while tissue mineral density was lower in the cortical compartment. Collagen crosslink ratio was higher in both cortical and trabecular compartments of male cKO mice. Our study suggested that BMP signaling in osteoblast mediated by BMP receptors, namely ACVR1 and BMPR1A, is critical in regulating bone quantity and bone quality.     

HLA-G gene polymorphism in a Japanese population

 Polymorphism of the HLA-G gene in a Japanese population was investigated employing polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis, PCR sequence-specific oligonucleotide (SSO) analysis, and DNA direct sequencing. Nucleotide sequence variations in exons 2, 3, and 4 of the HLA-G gene in 54 healthy Japanese individuals were examined. In addition, seven Japanese samples carrying common HLA haplotypes were analyzed. In total, nine single-base substitutions compared with the sequence of G ^* 01011 were identified: one in intron 1 (nucleotide position 970), one in exon 2 (the third base of codon 57: G → A), three in intron 2 (1264, 1276, and 1292), three in exon 3 (the third base of codon 93: C → T, the third base of codon 107: A → T, and the first base of codon 110: C → A), and one in intron 3 (2334). The substitution at codon 110 was non-synonymous and led to an amino acid substitution from leucine to isoleucine. The other three nucleotide substitutions in exons were synonymous. Through analysis of combinations of the exon 2, 3, and 4 nucleotide sequences we identified four alleles, which we provisionally designated GJ1, GJ2, GJ3, and GJ4. The allele frequencies were estimated to be 0.33, 0.16, 0.45, and 0.06, respectively. Nucleotide sequences of GJ1, GJ2, and GJ4 were identical to G ^* 01011, the clone 7.0E, and G ^* 01013, respectively. GJ3 was a newly observed allele and was officially designated G ^* 0104 by the WHO Nomenclature Committee in January 1996. Strong positive associations were observed between HLA-G alleles and HLA-A, -B, or -DRB1 alleles. Received: 15 February 1996 / Revised: 26 March 1996     

Dielectric study of the cooperative order–disorder transition in aqueous solutions of schizophyllan,a triple-helical polysaccharide

Schizophyllan exists in aqueous solution as a triple helix, which is intact at room temperature. Its aqueous solution forms some ordered structure at low temperatures but undergoes a sharp transition to a disordered structure as the temperature is raised. The transition temperature T_c is about 7 and 18°C for H₂O and D₂O solutions, respectively. This transition was followed by time-domain reflectometry to investigate dynamic aspects of the transition. In addition to a major peak around 10 GHz, the dielectric dispersion curve of a 20 wt % schizophyllan in D₂O exhibited a small peak around 100 MHz below T_c and around 10 MHz above T_c. The major peak is due to bulk water, whereas the 100 MH_z peak is assigned to “bound” or “structured” water, and that around 10 MHz to side-chain glucose residues. However, unlike usual bound water reported for biopolymer solutions, this “structured” water disappears abruptly when the temperature becomes close to T_c without accompanying a conformational transition of the main chain. The above assignment is consistent with the structure of the ordered phase derived from previous static data that it consists of side-chain glucose residues along with nearby water molecules surrounding the helix core that are interacting with each other loosely through hydrogen bonds, and spreads radially only a layer of one or two water molecules but a long distance along the helix axis. © 1995 John Wiley & Sons, Inc.     

The breakpoint on 7p in a patient with t(6;7) and craniosynostosis is spanned by a YAC clone containing the D7S503 locus

We previously reported a patient with an apparently balanced t(6;7) translocation and craniosynostosis. We now demonstrate, by fluorescence in situ hybridization, that the yeast artificial chromosome clone 933_e_l from the Centre d'Etude du Polymorphisme Humain library harbouring the D7S503 locus spans the breakpoint on distal 7p. Recent reports have defined a candidate region for a Saethre-Chotzen craniosynostosis locus between the loci D7S513 and D7S516, a region that includes the D7S503 locus. Since the translocation carrier shows only some of the symptoms characteristic for the Saethre-Chotzen syndrome, it remains unresolved whether the gene disrupted by the translocation event is the only one causing craniosynostosis in this chromosomal region.     

Circadian modulation in photic induction of Fos-like immunoreactivity in the suprachiasmatic nucleus cells of diurnal chipmunk,Eutamias asiaticus

Photic induction of immediate early genes including c-fos in the suprachiasmatic nucleus (SCN) has been well demonstrated in the nocturnal rodents. On the other hand, in diurnal rodents, no data is available whether the light can induce c-fos or Fos in the SCN. We therefore examined whether 60 min light exposure induces Fos-like immunoreactivity (Fos-lir) in the SCN cells of diurnal chipmunks and whether the induction is phase dependent, comparing with the results in nocturnal hamsters. We also examined an effect of light on the locomotor activity rhythm under continuous darkness. Fos-lir was induced in the chipmunk SCN. The induction was clearly phase dependent. The light during the subjective night induced strong expression of Fos-lir. This phase dependency is similar to that in hamsters. However, unlike in hamsters, the Fos-lir was induced in some SCN cells of chipmunks exposed to light during the subjective day. In the locomotor rhythm, on the other hand, the light pulse failed to induce the phase shift at phases at which the Fos-lir was induced. These results suggest that the photic induction of Fos-lir in the diurnal chipmunks is gated by a circadian oscillator as well as in the nocturnal hamsters. However, the functional role of Fos protein may be different in the diurnal rodents from in the nocturnal rodents.