Yoshiki Ohba

Recognition of phosphatidylserine on the surface of apoptotic spermatogenic cells and subsequent phagocytosis by Sertoli cells of the rat.

In a primary co-culture of spermatogenic and Sertoli cells of the rat, many spermatogenic cells die by apoptosis and are subsequently engulfed by Sertoli cells. We investigated the mechanism of this phagocytosis reaction. Testicular cells from 20-day-old rats were cultured, and spermatogenic cells and Sertoli cells were separated. When the recovered spermatogenic cells were maintained without Sertoli cells, the viability of the cells decreased and they became more susceptible to phagocytosis by Sertoli cells. Phagocytosis was severely impaired when liposomes containing acidic phospholipids, such as phosphatidylserine, phosphatidylinositol, and cardiolipin, were included in the reaction, whereas those consisting of neutral phospholipids showed little effect. Such anionic liposomes were more efficiently engulfed by Sertoli cells than were the other neutral liposomes. Also, the number of spermatogenic cells that exposed phosphatidylserine to the surface increased when cells were maintained in single culture. The results indicate that upon induction of spermatogenic cell apoptosis, phosphatidylserine and probably other acidic phospholipids, which are normally localized in the inner leaflet of the plasma membrane, translocate to the outer leaflet and serve as a signal for phagocytosis by Sertoli cells.

Dephosphorylation of human p34cdc2 kinase on both Thr-14 and Tyr-15 by human cdc25B phosphatase.

In mammalian cells, p34 cdc2 kinase undergoes phosphorylation at threonine‐14, tyrosine‐15 and threonine‐161 in the S and G2 phases of the cell cycle. At the onset of mitosis, the kinase becomes dephosphorylated at threonine‐14 and tyrosine‐15, resulting in activation. Cdc25 phosphatase has been shown to dephosphorylate tyrosine‐15 in vitro, but whether it also does at threonine‐14 remains unclear. In this study, we have found that human cdc25B phosphatase dephosphorylates both threonine‐14 and tyrosine‐15 but not threonine‐161.

Structure of nucleohistone: I. Hydrodynamic behaviour

Abstract Partially dehistonized nucleohistone was prepared by a Sephadex G-100 gel filtration method and its hydrodynamic properties were examined by measuring its flow birefringence, flow dichroism and sedimentation coefficient. On association with histones the apparent length of DNA molecules decreased, and the number of base pairs perpendicular to the axis of the molecule decreased by 60 %. These configurational distortions were considered to be largely due to arginine-rich histones in the molecule. The heterogeneous components of histones were almost uniformly distributed throughout the molecular population of the nucleohistones.

In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase

The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.

CO-EXPRESSION OF FAS AND FAS-LIGAND ON THE SURFACE OF INFLUENZA VIRUS-INFECTED CELLS

Influenza virus-infected cultured cells undergo apoptosis after an increment of Fas (APO-1/CD95) on the cell surface. By flow cytometry, cell surface Fas-ligand was detected in virus-infected cells with a time course similar to that of Fas. Moreover, Fas and Fas-ligand were co-expressed in those cells. The mode of induction, however, appeared to be distinct for the two proteins. Influenza virus infection induced the externalization of phosphatidylserine on the cell surface at the early stage of apoptosis, an event that has been observed in cells undergoing Fas-mediated apoptosis. In fact, apoptosis of the virus-infected cells was inhibited in the presence of an antagonistic anti-Fas-ligand monoclonal antibody. These results suggest that influenza virus infection causes augmented expression of both Fas and Fas-ligand and apoptosis is induced when the infected cells come into contact with each other.

Structure of nucleohistone. II. Thermal denaturation.

Abstract The spectral change accompanying the thermal denaturation of calf-thymus nucleohistone was resolved into characteristics of the disruption of the adenine thymine (A-T) and guanine-cytosine (G-C) pairs in the DNA as a function of the temperature. It was shown that in nucleohistone, as distinct from DNA, the G-C pairs are more susceptible to thermal denaturation than the A-T pairs. The stabilization of the A-T pairs was attributed to the lysine residues in the histones.

Microtubule destabilization by cdc2/H1 histone kinase: Phosphorylation of a “Pro-rich region” in the microtubule-binding domain of MAP-4

Microtubule-associated protein-4 (MAP-4), a major MAP in proliferating cells, consists of a microtubule-binding domain and a projection domain protruding from the microtubule wall. The former contains a Pro-rich region and an assembly-promoting (AP) sequence region which is common to the neuron-specific MAPs, MAP-2 and tau1. In this paper, we describe the phosphorylation of the Pro-rich region of MAP-4 and the suppression of its assembly-promoting activity by cdc2/H1 histone kinase. This inactivation of MAP-4 may cause disassembly of the interphase microtubular network at the end of the G2 phase of the cell cycle.

The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase

The human weel protein, a homologue of the yeast weel protein, was expressed in E. coli and purified to homogeneity. The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1. It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo. Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not. These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase.

A theoretical consideration of the abnormal behavior of histones on sodium dodecylsulfate gel electrophoresis

Several proteins were subjected to sodium dodecylsulfate gel electrophoresis at various concentrations of gel, and the logarithm of their relative mobilities (RF) versus gel concentrations (T) were plotted (Ferguson plot). This shows that their mobilities are all the same in free solution (RF0) and that the slopes in the Ferguson plot (K′R) are linearly related to their molecular weights (Mr). These results mean that log RF versus Mr, log Mr versus RF′ and K′R versus Mr plots give straight lines over a considerably wide range of Mr values. Histones, however, show abnormal behavior in the log RF versus Mr plot or the log Mr versus RF plot at a fixed gel concentration, although in the K′R versus Mr plot they all fall on the line of ordinary proteins. These phenomena about histones were found to be attributable to the unusually low RF0 values as compared with the ordinary proteins which means that the log RF versus Mr plot or the log Mr versus RF plot at a given gel concentration are unsuitable for the molecular weight estimation of basic proteins like histones, but the K′R versus Mr plot should be adopted. A Ferguson plot of the histones revealed that at a gel concentration between 11 to 14%, all the five histones are resolved, the best resolution was obtained at 12.5% gel.

Mouse p87 wee1 kinase is regulated by M-phase specific phosphorylation

We have cloned a mousewee1 kinase cDNA (mwee1). The clone is 2258 bp in length and its open reading frame corresponds to 646 amino acid residues. The molecular weight of this kinase is 87 kDa in SDS-PAGE, which is about 1.7-fold larger than the human p50wee1 kinase reported previously. In a cell cycle, the mousewee1 kinase is phosphorylated at M-phase, and anin vitro study using a mitotic extract revealed that phosphorylation occurs in the N-terminal domain, which is absent from the humanwee1 kinase, resulting in inactivation of the kinase activity. The N-terminal domain or entire molecule is extensively phosphorylated bycdc2-cyclin B kinase. Furthermore, the activity of thewee1 kinase was reduced by phosphorylation with the mitotic extract which containedcdc2-cyclin B kinase