Clinical and molecular studies in 15 females with ring X chromosomes: implications for r(X) formation and mental development

We report clinical and molecular findings in 15 Japanese mosaic females with r(X) chromosomes, 45,X/46,X,r(X), confirmed by fluorescence in situ hybridization (FISH) analysis for DXZ1 and whole X chromosome painting. Cases 1-3, 5-7, and 11-13 had mental retardation (MR), the remaining cases being free from MR. FISH analysis showed that XIST was absent from the r(X) chromosomes in cases 1-4 and was present on the r(X) chromosomes in cases 5-15. X-inactivation analysis for the methylation status of the AR gene indicated that, of eight cases with XIST-positive r(X) chromosomes in more than 10% (23%-62%) of lymphocytes (cases 5-12), cases 5-10 had selective X-inactivation, whereas cases 11 and 12 had active X disomy. Microsatellite analysis for multiple loci on the pericentromeric region revealed that, of 11 cases with r(X) chromosomes in more than 10% (13%-62%) of lymphocytes (cases 1, 2, and 4-12), cases 1, 2, and 5-10 had heterozygous alleles for at least one locus, whereas cases 4, 11, and 12 had single alleles for all the loci examined. The results suggest that the r(X) and normal X chromosomes could be of biparental or uniparental origin, and that mental status in females with r(X) chromosomes is determined by multiple factors, including the presence or absence of XIST on the r(X) chromosomes and the size and frequency of active r(X) chromosomes, in addition to co-incidental genetic and environmental factors.     

In Vivo Evaluation of Hippocampal Anti-Oxidant Ability of Zonisamide in Rats

We evaluated the anti-oxidant property of zonisamide (ZNS) in the rat brain under freely moving conditions by means of in vivo microdialysis of two exogenous nitroxide radicals, 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (carbamoyl-PROXYL) and 3-methoxy carbonyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (PCAM). Time-dependent changes in the signal intensities of these exogenous nitroxide radicals obtained from the hippocampal perfusates were observed using an X-band ESR spectrometer at 20-min intervals. The ESR signal intensities of nitroxide radicals decreased exponentially in all animals, which indicates that their half-life could be used as a parameter to estimate the decay rate of nitroxide radicals. Nitroxide radicals lose their paramagnetism when exposed to reductants in a biological system. Thus, half-life reflects the in vivo reducing ability. Although the half-life of carbamoyl-PROXYL, which could not pass the blood-brain barrier (BBB), was not changed when compared with the controls, pre-treatment with ZNS significantly shortened the half-life of PCAM, which could pass through the BBB. These findings suggest that the ZNS-induced increase in reducing ability did not occur within the extracellular space, but rather mainly at the neural cell membrane. This study is the first in vivo evaluation of the reducing ability of ZNS in freely moving animals.     

Morphogenesis in cucumber seedlings is negatively controlled by gravity

 Seedlings of most cucurbitaceous plants develop a peg (protuberance caused by cell outgrowth) on the transition zone between the hypocotyl and root. The peg is necessary for removing the seed coat after germination. In our spaceflight experiments on the STS-95 space shuttle, Discovery, we found that cucumber (Cucumis sativus L.) seedlings grown under microgravity conditions developed two pegs symmetrically at the transition zone. Thus, cucumber seedlings potentially develop two pegs and do not require gravity for peg formation itself, but on the ground the development of one peg is suppressed in response to gravity. This may be considered as negative control of morphogenesis by gravity. Received: 17 August 1999 / Accepted: 4 October 1999     

Ligand-induced conformational changes via flexible linkers in the amino-terminal region of the inositol 1,4,5-trisphosphate receptor

Cytoplasmic Ca2+ signals are highly regulated by various ion transporters, including the inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R), which functions as a Ca2+ release channel on the endoplasmic reticulum membrane. Crystal structures of the two N-terminal regulatory regions from type 1 IP(3)R have been reported; those of the IP(3)-binding core (IP(3)R(CORE)) with bound IP(3), and the suppressor domain. This study examines the structural effects of ligand binding on an IP(3)R construct, designated IP(3)R(N), that contains both the IP(3)-binding core and the suppressor domain. Our circular dichroism results reveal that the IP(3)-bound and IP(3)-free states have similar secondary structure content, consistent with preservation of the overall fold within the individual domains. Thermal denaturation data show that, while IP(3) has a large effect on the stability of IP(3)R(CORE), it has little effect on IP(3)R(N), indicating that the suppressor domain is critical to the stability of IP(3)R(N). The NMR data for IP(3)R(N) provide evidence for chemical exchange, which may be due to protein conformational dynamics in both apo and IP(3)-bound states: a conclusion supported by the small-angle X-ray scattering data. Further, the scattering data show that IP(3)R(N) undergoes a change in average conformation in response to IP(3) binding and the presence of Ca2+ in the solution. Taken together, these data lead us to propose that there are two flexible linkers in the N-terminal region of IP(3)R that join stably folded domains and give rise to an equilibrium mixture of conformational sub-states containing compact and more extended structures. IP(3) binding drives the conformational equilibrium toward more compact structures, while the presence of Ca2+ drives it to a more extended set.     

Novel action of apolipoprotein E (ApoE): ApoE isoform specifically inhibits lipid-particle-mediated cholesterol release from neurons

Background

Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular Aβ and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils.

Results

We immunized rabbits with a morphologically homogeneous population of Aβ42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 × G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils.

Conclusion

Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.     

Analysis of modified apolipoprotein B-100 structures formed in oxidized low-density lipoprotein using LC-MS/MS

Oxidatively modified low-density lipoprotein (oxLDL) is one of the major factors involved in the development of atherosclerosis. Because of the insolubility of apolipoprotein B-100 (apoB-100) and the heterogeneous nature of oxidative modification, modified structures of apoB-100 in oxLDL are poorly understood. We applied an on-Membrane sample preparation procedure for LC-MS/MS analysis of apoB-100 proteins in native and modified low-density lipoprotein (LDL) samples to eliminate lipid components in the LDLs followed by collection of tryptic digests of apoB-100. Compared with a commonly used in-gel digestion protocol, the sample preparation procedure using PVDF membrane greatly increased the recovery of tryptic peptides and resulted in improved sequence coverage in the final analysis, which lead to the identification of modified amino acid residues in copper-induced oxLDL. A histidine residue modified by 4-hydroxynonenal, a major lipid peroxidation product, as well as oxidized histidine and tryptophan residues were detected. LC-MS/MS in combination with the on-Membrane sample preparation procedure is a useful method to analyze highly hydrophobic proteins such as apoB-100.     

Midostaurin preferentially attenuates proliferation of triple-negative breast cancer cell lines through inhibition of Aurora kinase family

Background

Breast cancer is classified into three subtypes by the expression of biomarker receptors such as hormone receptors and human epidermal growth factor receptor 2. Triple-negative breast cancer (TNBC) expresses none of these receptors and has an aggressive phenotype with a poor prognosis, which is insensitive to the drugs that target the hormone receptors and human epidermal growth factor receptor 2. It is, thus, required to develop an effective therapeutic reagent to treat TNBC.

Results

The study using a panel of 19 breast cancer cell lines revealed that midostaurin, a multi-target protein kinase inhibitor, suppresses preferentially the growth of TNBC cells comparing with non-TNBC cells. Clustering analysis of the drug activity data for the panel of cancer cell lines predicted that midostaurin shares the target with Aurora kinase inhibitors. Following studies indicated that midostaurin attenuates the phosphorylation reaction mediated by Aurora kinase in the cells and directly inhibits this protein kinase in vitro, and that this reagent induces apoptosis accompanying accumulation of 4N and 8N DNA cells in TNBC cells.

Conclusion

Midostaurin suppresses the proliferation of TNBC cells among the breast cancer cell lines presumably through the inhibition of the Aurora kinase family. The precise study of midostaurin on cell growth will contribute to the development of the drug for the treatment of TNBC.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0150-2) contains supplementary material, which is available to authorized users.     

Ongoing localized extinctions of stream-dwelling white-spotted charr populations in small dammed-off habitats of Hokkaido Island,Japan

Habitat fragmentation caused by damming can greatly reduce the population viability of aquatic organisms, with smaller fragmented populations at higher risk of extinction due to increased demographic, genetic, and environmental stochasticity. However, empirical evidence demonstrating that smaller natural populations are more vulnerable to extinction is limited. We studied the vulnerability to extinction of white-spotted charr (Salvelinus leucomaenis) populations in 30 dammed-off streams in Oshima Peninsula, southwestern Hokkaido Island, Japan, by comparing the incidence of charr populations in streams between 1999 and 2014. Using electrofishing and environmental DNA surveys, we identified three localized extinctions, with the probability of extinction increasing with decreasing watershed area (our surrogate for habitat size). We also found a new population in one dammed-off stream in which white-spotted charr were previously unknown, after installation of a fish ladder, indicating the capacity of white-spotted charr to recolonize reconnected habitat in a short period. Our results suggest that localized extinction of white-spotted charr in small dammed-off streams is ongoing, but that appropriate fish migration corridors can reduce localized extinction risk and increase the probability of species persistence.