Silencing of p53 and CDKN1A establishes sustainable immortalized megakaryocyte progenitor cells from human iPSCs.

Platelet transfusions are critical for severe thrombocytopenia but depend on blood donors. The shortage of donors and the potential of universal HLA-null platelet products have stimulated research on the ex vivo differentiation of human pluripotent stem cells (hPSCs) to platelets. We recently established expandable immortalized megakaryocyte cell lines (imMKCLs) from hPSCs by transducing MYC, BMI1, and BCL-XL (MBX). imMKCLs can act as cryopreservable master cells to supply platelet concentrates. However, the proliferation rates of the imMKCLs vary with the starting hPSC clone. In this study, we reveal from the gene expression profiles of several MKCL clones that the proliferation arrest is correlated with the expression levels of specific cyclin-dependent kinase inhibitors. Silencing CDKN1A and p53 with the overexpression of MBX was effective at stably inducing imMKCLs that generate functional platelets irrespective of the hPSC clone. Collectively, this improvement in generating imMKCLs should contribute to platelet industrialization and platelet biology.

Reprofiling a classical anthelmintic, pyrvinium pamoate, as an anti-cancer drug targeting mitochondrial respiration.

Pyrvinium pamoate (PP) is an FDA-approved classical anthelmintic, but is now attracting particular attention as an anti-cancer drug after recent findings of its potent cytotoxicity against various cancer cell lines only during glucose starvation, as well as its anti-tumor activity against hypovascular pancreatic cancer cells transplanted in mice. The molecular mechanisms by which PP promotes such preferential toxicity against cancer cells are currently under extensive investigation. PP suppressed the NADH-fumarate reductase system that mediates a reverse reaction of the mitochondrial electron-transport chain complex II in anaerobic organisms such as parasitic helminthes or mammalian cells under tumor microenvironment-mimicking hypoglycemic/hypoxic conditions, thereby inhibiting efficient ATP production. PP also inhibited the unfolded protein response induced by glucose starvation, thereby inhibiting the proliferation of pancreatic cancer cells. Even under normoglycemic/normoxic conditions, PP suppressed the mitochondrial electron-transport chain complex I and thereby STAT3, inhibiting the proliferation of myeloma/erythroleukemia cells. Here, we review accumulating knowledge on its working mechanisms and evaluate PP as a novel anti-cancer drug that targets mitochondrial respiration.

Pyrvinium pamoate inhibits proliferation of myeloma/erythroleukemia cells by suppressing mitochondrial respiratory complex I and STAT3.

Pyrvinium pamoate (PP), a classical anthelminthic, potently inhibited proliferation and STAT3 Tyr705 phosphorylation of human myeloma (U266B1 and PCM6)/erythroleukemia (HEL 92.1.7) cells. PCM6 cell proliferation was markedly impaired by STAT3 siRNA knockdown. PP inhibited ATP production/O(2) consumption in those three cells and mitochondrial respiratory complex (I+III, but not II+III) activity in mouse kidney mitochondrial fractions. PP inhibition of ATP production, STAT3 Tyr705 phosphorylation, and proliferation was absent in mitochondrial DNA-deficient HEL 92.1.7-ρ(0) cells. Moreover, PP acted synergistically with dexamethasone to inhibit PCM6 cell proliferation. In conclusion, we identified PP as a potential anticancer drug directed against mitochondrial respiratory complex I/STAT3.

Comparative study on the morphology and the composition of the otoliths in the teleosts.

CONCLUSION: Saccular otoliths of teleosts were mostly larger than utricular otoliths, which might relate to the three-dimensional movement. The large and heavy otolith may be better suited in saccules of the bottom and reef fishes. The quantities of iron in lagenar otoliths were found to be lower than those of birds. The function of the fish lagena remains to be elucidated by further studies. OBJECTIVE: To evaluate the morphological characteristics and the chemical composition of the otoliths in fishes as related to behaviour and habitat. MATERIALS AND METHODS: We studied the morphology of the otoliths of 18 genera of fishes (81 samples) divided into 3 groups: saltwater fish (13 genera), freshwater fish except for the carp family (3 genera) and carp family fish (2 genera). The otoliths and the living environments were compared. The chemical composition was analysed using a synchrotron X-ray fluorescence analyser. RESULTS: Bottom fishes generally have larger saccular otoliths, and migrating fishes have smaller saccular otoliths. In comparing the bottom/reef fishes and the migrating fishes in salt water, the former tended to have larger saccular otoliths. In saltwater bottom fishes the tendency was found that the thinner the head, the larger was the saccular otolith. We found significant quantities of iron, zinc and manganese in the lagenar otoliths.

The relation between the migration function of birds and fishes and their lagenal function.

CONCLUSION: The lagena of pigeons is a unique organ and it is concluded that it is a key element in the magnetic sensor system of pigeons and migrating birds. The lagenal otolith in pigeons contains more iron than saccular and utricular otoliths. The function of the lagena of pigeons was clarified because the homing ability of pigeons was largely disrupted after unilateral lagenal nerve section and attachment of magnetic balls with a magnetic field strength under 5 Gauss. The lagena of pigeons may have a navigational function as a geomagnetic sensor. OBJECTIVE: Otoliths of many kinds of fishes and birds were analyzed. MATERIALS AND METHODS: The otoliths of fish and birds were analyzed using synchrotron X-ray fluorescence analysis. Behavioral experiments concerning homing ability of pigeons were done by sectioning their lagenal nerves or interfering with the function of the lagena using a magnet. Twenty-one birds were treated in this way and 30 birds from the same loft of racing pigeons were used as controls. RESULTS: By comparing the compositions of the three different kinds of otoliths among several species of sea fish and birds, it was found that the saccular and utricular otoliths contain scarcely detectable levels of iron but that iron is present in significant quantities in the lagenal otoliths of the birds and sea fish. The results of homing tests clearly revealed a magnetic influence on the function of the lagena in terms of navigation ability of pigeons. The treated pigeons were either lost or significantly delayed while the controls returned within 30 min of release.

CD41+/CD45+ cells without acetylcholinesterase activity are immature and a major megakaryocytic population in murine bone marrow.

Murine megakaryocytes (MKs) are defined by CD41/CD61 expression and acetylcholinesterase (AChE) activity; however, their stages of differentiation in bone marrow (BM) have not been fully elucidated. In murine lineage-negative (Lin(-))/CD45(+) BM cells, we found CD41(+) MKs without AChE activity (AChE(-)) except for CD41(++) MKs with AChE activity (AChE(+)), in which CD61 expression was similar to their CD41 level. Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs could differentiate into AChE(+), with an accompanying increase in CD41/CD61 during in vitro culture. Both proplatelet formation (PPF) and platelet (PLT) production for Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs were observed later than for Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs, whereas MK progenitors were scarcely detected in both subpopulations. GeneChip and semiquantitative polymerase chain reaction analyses revealed that the Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are assigned at the stage between the progenitor and PPF preparation phases in respect to the many MK/PLT-specific gene expressions, including beta1-tubulin. In normal mice, the number of Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs was 100 times higher than that of AChE(+) MKs in BM. When MK destruction and consequent thrombocytopenia were caused by an antitumor agent, mitomycin-C, Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs led to an increase in AChE(+) MKs and subsequent PLT recovery with interleukin-11 administration. It was concluded that MKs in murine BM at least in part consist of immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs and more differentiated Lin(-)/CD41(++)/CD45(+)/AChE(+) MKs. Immature Lin(-)/CD41(+)/CD45(+)/AChE(-) MKs are a major MK population compared with AChE(+) MKs in BM and play an important role in rapid PLT recovery in vivo.

Efficient gene expression in megakaryocytic cell line using nucleofection.

To clarify the mechanism of platelet production from megakaryocytes, expression of target proteins by gene transfection was examined using various gene delivery techniques. Transfection into hematopoietic cells, including megakaryocytes, by conventional gene delivery techniques such as electroporation and lipofection are known to be difficult. In this study, in addition to electroporation and lipofection, we tested other gene-transfer methods (nucleofection, transfection using inactivated virus envelope, and transferrin-linked cationic polymer) with the green fluorescent protein (GFP) gene into the human megakaryocytic cell line MEG-01. We found that nucleofection, which uses a combination of special electrical parameters and specific solutions, was the best, judging from the expression ratio of GFP-positive cells (approximately 70% of cells) and low toxicity. The efficiency of GFP expression was not related to the amount of pDNA delivered into the MEG-01 cells. To verify the utility of nucleofection, the thrombopoietin (TPO) receptor c-mpl was transfected into MEG-01 cells. Transfected cells showed a higher responsiveness to TPO than mock-transfected MEG-01 cells. We propose that nucleofection is a useful method for transfecting target genes to megakaryocytic cells when addressing the mechanism of platelet production.

Constituents of holothuroidea, 12. Isolation and structure of glucocerebrosides from the sea cucumber Holothuria pervicax.

Ten glucocerebrosides, HPC-3-A-HPC-3-J, have been isolated from their obtained parent glucocerebroside molecular species HPC-3, together with other glucocerebroside molecular species HPC-1 and HPC-2, from the less polar lipid fraction of a chloroform/methanol extract of the sea cucumber Holothuria pervicax. The structures of these glucocerebrosides have been determined on the basis of chemical and spectroscopic evidence. Reversed-phase HPLC, including a recycling system, was effective in isolating these glucocerebrosides, revealing a very close resemblance in structure, though the problem due to regio-isomers remains.

Comparison of the morphology of the inner ear between newts and frogs in relation to their locomotory capability.

The ultrastructural differences between the inner ears of Japanese red-bellied newts (Cynops pyrrhogaster) and black-spotted pond frogs (Rana nigromaculata) were investigated. Scanning electron microscopic observations showed apparent morphological differences in the shape of the ampulla cristae and the localization of the striola in the saccular macula. There were differences in the length of the kinocilia of the sensory hairs in each sensory region. In addition, the diameters of the bundles of stereocilia differed between the two species: the bundles of stereocilia in the semicircular cristae were thicker in frogs than in newts, while those of the utricular and lagenal maculae were thicker in newts than in frogs.

Experimental analysis of behavior of homing pigeons as a result of functional disorders of their lagena.

Behavioral experiments concerning the homing abilities of pigeons were done by sectioning their lagenal nerves or interfering with the function of the lagena using a magnet. Twenty-one birds were treated in this way and 30 birds from the same loft of racing pigeons were used as controls. The results of homing tests clearly revealed a magnetic influence on the function of the lagena in terms of the navigation ability of pigeons: the treated birds were either lost or significantly delayed while the controls returned within 30 min of release. The lagena of birds is a unique organ and it is concluded that it is a key element in the magnetic sensory system of birds.