The telomerase activity and length of telomeres of peripheral blood mononuclear cells obtained from 124 healthy individuals aged 4-95 years was measured. Telomerase activity level was semiquantitatively assessed by a fluorescent-telomeric repeat amplification protocol (fluorescent-TRAP) using an internal telomerase assay standard, fluorescent primers and an automated laser fluorescent DNA sequencer. Telomeric length, measured by assay of terminal restriction fragments (TRFs), was determined in HinfI-digested DNA by Southern blot analysis using a (TTAGGG)4 probe. TRF length was determined in 80 individuals and age-related progressive reduction of size was observed. TRF length in peripheral blood mononuclear cells obtained from normal individuals (aged 4-39 years) decreased by approximately 84 bp per year, while in individuals aged > or = 40 years it decreased by 41 bp per year. In contrast, telomerase activity showed an apparent biphasic pattern with aging. Individuals aged 4-39 years showed a progressive decrease in telomerase activity, whereas 65% of those aged > or = 40 years showed relatively stable but very low telomerase activity, and the remaining individuals aged > or = 40 years had no detectable telomerase activity. These data obtained from normal individuals might in the future be of value to help risk stratify and manage the care of patients with leukemia.

Immunolocalization of 14-3-3 proteins in Alzheimer's disease (AD) brains was investigated using isoform-specific antibodies. Weak granular immunoreactivity of 14-3-3 proteins was found in neuronal cytoplasm in control subjects and AD brains. Both intracellular and extracellular neurofibrillary tangles (NFTs), as well as neuropil thread-like structures, were immunopositive for 14-3-3 proteins. This was corroborated by triple-fluorolabeling method visualizing paired helical filament (PHF) tau and 14-3-3 epitopes in relation to fibrillary state detected by thiazin red. Pretangle neurons (positive for PHF-tau without fibrillary structure detected by thiazin red) only contained fine granular immunoreactivity (IR) of 14-3-3, which was similarly found in unaffected neurons. Granular cytoplasmic IR of 14-3-3 proteins in pretangle neurons was not colocalized to granular tau-like IR, which suggests that participation of 14-3-3 proteins in NFT formation was restricted to its later stages. Its zeta isoform was most prominent in these NFTs, suggesting that this isoform is a major component involved in the formation of NFTs. In contrast, IR of epsilon isoform was found in the neuropil of the hippocampus and that of sigma isoform was localized to granule cells of the dentate gyrus in AD brains, as seen in the age-matched controls. Expression of 14-3-3 proteins were found to be highly variable and dependent on their isoforms, regions and cell types. Molecular, as well as topographical, dissection of 14-3-3 proteins will provide us with an improved understanding of this molecule in normal and pathological conditions.

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An activating mutation of () is the most frequent genetic alteration associated with poor prognosis in acute myeloid leukemia (AML). Although many FLT3 inhibitors have been clinically developed, no first-generation inhibitors have demonstrated clinical efficacy by monotherapy, due to poor pharmacokinetics or unfavorable safety profiles possibly associated with low selectivity against FLT3 kinase. Recently, a selective FLT3 inhibitor, quizartinib, demonstrated favorable outcomes in clinical studies. However, several resistant mutations emerged during the disease progression. To overcome these problems, we developed a novel FLT3 inhibitor, FF-10101, designed to possess selective and irreversible FLT3 inhibition. The co-crystal structure of FLT3 protein bound to FF-10101 revealed the formation of a covalent bond between FF-10101 and the cysteine residue at 695 of FLT3. The unique binding brought high selectivity and inhibitory activity against FLT3 kinase. FF-10101 showed potent growth inhibitory effects on human AML cell lines harboring internal tandem duplication (-ITD), MOLM-13, MOLM-14, and MV4-11, and all tested types of mutant FLT3-expressing 32D cells including quizartinib-resistant mutations at D835, Y842, and F691 residues in the FLT3 kinase domain. In mouse subcutaneous implantation models, orally administered FF-10101 showed significant growth inhibitory effect on FLT3-ITD-D835Y- and FLT3-ITD-F691L-expressing 32D cells. Furthermore, FF-10101 potently inhibited growth of primary AML cells harboring either -ITD or-D835 mutation in vitro and in vivo. These results indicate that FF-10101 is a promising agent for the treatment of patients with AML with mutations, including the activation loop mutations clinically identified as quizartinib-resistant mutations.

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Platelets were activated in the cerebral circulation of patients with stroke even in the chronic phase, which suggests the development of underlying vascular lesions and of thrombogenesis with or without infarction. Platelets were activated mainly within the heart in cases of cardioembolic stroke. An enhanced release reaction secondary to platelet activation was often seen in patients with Binswanger's disease, which indicates that its pathophysiology differs from that of other subtypes of stroke.

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