Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus: modulation by cellular immortalization and senescence

B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus have a phenotype corresponding to activated B-lymphoblasts. Although they are widely used as models in various biological and medical studies, their innate morphological differentiation and apoptosis has been little studied. We report here that a large proportion of LCL cells spontaneously differentiate into smaller lymphoid cells which ultimately undergo apoptosis during conventional cell culture. Two distinct types of apoptosis with some intermediate types exist: type 1 apoptosis in small and medium-size cells with shrunken nuclei having heavily condensed chromatin in the whole nucleus region accompanied by relatively large internucleosomally fragmented DNA (above 2 kbp); type 2 apoptosis in large lymphoblasts with extremely lobulated nuclei having chromatin condensation beneath the nuclear membrane alone accompanied by smaller internucleosomally fragmented DNA (below 2 kbp). Type 1 apoptotic cells were far more numerous than type 2 apoptotic cells. The incidence of type 1 apoptosis was suppressed by cellular immortalization and was extremely stimulated at the end of the lifespan (crisis). These results provide essential information for us to use LCLs for various biological and medical studies including cellular immortalization, tumorigenesis and senescence.     

The p250GAP gene is associated with risk for schizophrenia and schizotypal personality traits

Background

Hypofunction of the glutamate N-Methyl-d-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. p250GAP is a brain-enriched NMDA receptor-interacting RhoGAP. p250GAP is involved in spine morphology, and spine morphology has been shown to be altered in the post-mortem brains of patients with schizophrenia. Schizotypal personality disorder has a strong familial relationship with schizophrenia. Several susceptibility genes for schizophrenia have been related to schizotypal traits.

Methods

We first investigated the association of eight linkage disequilibrium-tagging single-nucleotide polymorphisms (SNPs) that cover the p250GAP gene with schizophrenia in a Japanese sample of 431 schizophrenia patients and 572 controls. We then investigated the impact of the risk genetic variant in the p250GAP gene on schizotypal personality traits in 180 healthy subjects using the Schizotypal Personality Questionnaire.

Results

We found a significant difference in genotype frequency between the patients and the controls in rs2298599 (χ² = 17.6, p = 0.00015). The minor A/A genotype frequency of rs2298599 was higher in the patients (18%) than in the controls (9%) (χ² = 15.5, p = 0.000083). Moreover, we found that subjects with the rs2298599 risk A/A genotype, compared with G allele carriers, had higher scores of schizotypal traits (F_1,178 = 4.08, p = 0.045), particularly the interpersonal factor (F_1,178 = 5.85, p = 0.017).

Discussion

These results suggest that a genetic variation in the p250GAP gene might increase susceptibility not only for schizophrenia but also for schizotypal personality traits. We concluded that the p250GAP gene might be a new candidate gene for susceptibility to schizophrenia.     

Effect of Ionic Strength in the Gel Filtration of Charged Polysaccharides

ABSTRACT

Collagen-derived dipeptide prolyl hydroxyproline (Pro-Hyp) is involved in the proliferation and differentiation of various types of cultured cells. To elucidate the mechanism underlying Pro-Hyp actions during osteoblast differentiation, we hypothesized that proteins binding to Pro-Hyp serve to mediate cellular signaling, affecting Runx2 expression. Recently, we performed the characterization of Foxg1, that it enhances Runx2 expression in the presence of Pro-Hyp. Our findings indicate that Pro-Hyp directly binds to the Foxg1 recombinant protein, which leads to the structural alteration of the Foxg1 protein. In addition, Foxg1 appears to interact with Runx2 in the absence of Pro-Hyp, with Pro-Hyp disrupting the interaction between Foxg1 and Runx2. Collectively, our results indicate that the Pro-Hyp bound Foxg1 alters the structured conformation of Foxg1, resulting in conformational changes that lead to dissociation from Runx2. These novel findings suggest that during osteoblast differentiation, Pro-Hyp mediates Runx2 activity though directly binding to Foxg1 and increases Runx2 expression.

Abbreviations: CPT: collagen peptide; GST: Glutathione S-transferase; PAGE: Polyacrylamide gel electrophoresis; PCR: Polymerase chain reaction; prolyl hydroxyproline: Pro-Hyp     

Suppression of Coronary Atherosclerosis by Helix B Surface Peptide,a Nonerythropoietic,Tissue-Protective Compound Derived from Erythropoietin

Erythropoietin (EPO), a type I cytokine originally identified for its critical role in hematopoiesis, has been shown to have nonhematopoietic, tissue-protective effects, including suppression of atherosclerosis. However, prothrombotic effects of EPO hinder its potential clinical use in nonanemic patients. In the present study, we investigated the antiatherosclerotic effects of helix B surface peptide (HBSP), a nonerythropoietic, tissue-protective compound derived from EPO, by using human umbilical vein endothelial cells (HUVECs) and human monocytic THP-1 cells in vitro and Watanabe heritable hyperlipidemic spontaneous myocardial infarction (WHHLMI) rabbits in vivo. In HUVECs, HBSP inhibited apoptosis (≈70%) induced by C-reactive protein (CRP), a direct mediator of atherosclerosis. By using a small interfering RNA approach, Akt was shown to be a key molecule in HBSP-mediated prevention of apoptosis. HBSP also attenuated CRP-induced production of tumor necrosis factor (TNF)-α and matrix metalloproteinase-9 in THP-1 cells. In the WHHLMI rabbit, HBSP significantly suppressed progression of coronary atherosclerotic lesions as assessed by mean cross-sectional stenosis (HBSP 21.3 ± 2.2% versus control peptide 38.0 ± 2.7%) and inhibited coronary artery endothelial cell apoptosis with increased activation of Akt. Furthermore, TNF-α expression and the number of M1 macrophages and M1/M2 macrophage ratio in coronary atherosclerotic lesions were markedly reduced in HBSP-treated animals. In conclusion, these data demonstrate that HBSP suppresses coronary atherosclerosis, in part by inhibiting endothelial cell apoptosis through activation of Akt and in association with decreased TNF-α production and modified macrophage polarization in coronary atherosclerotic lesions. Because HBSP does not have the prothrombotic effects of EPO, our study may provide a novel therapeutic strategy that prevents progression of coronary artery disease.     

N-terminal mutational analysis of the interaction between growth-blocking peptide (GBP) and receptor of insect immune cells

GBP, a small insect cytokine isolated from lepidopterans, has a variety of functions. We constructed a series of mutants focusing on the unstructured N-terminal residues of GBP by acetylation, deletion, and elongation in order to investigate the interaction between GBP and its receptor in plasmatocytes. The 1H NMR spectra showed no significant changes in the tertiary structures of these peptides, which indicated that all the mutants maintained their core beta-sheet structures. The deletion and acetylated mutants, 2-25GBP, Ac2-25GBP, and AcGBP, lost their activity. 2-25GBP was the strongest antagonist, while Ac2-25GBP and AcGBP were moderate. In contrast, the elongated mutants, (-1R)GBP, (-1A)GBP, and (-2G,-1R)GBP maintained their plasmatocyte-spreading activity. These results demonstrate the importance of the GBP N-terminal charged amine and length of N-terminal GBP-peptide backbone for plasmatocyte-spreading activity. Next, we analyzed other mutant peptides, 1-25(N2A)GBP and 2-25(N2A)GBP, focusing on Asn2. Surprisingly, 2-25(N2A)GBP had slight plasmatocyte-spreading activity, whereas 2-25GBP lost its activity. Finally, substituted mutant, F3AGBP, had neither plasmatocyte-spreading activity nor antagonistic activity. These results demonstrate the function of each N-terminal residue in the interaction between GBP and its receptor in plasmatocytes.     

Effect of taurine on cholesterol metabolism in hamsters: up-regulation of low density lipoprotein (LDL) receptor by taurine

The effects of taurine on hepatic cholesterol metabolism were investigated in hamsters fed a high-fat diet or normal chow. Two weeks-treatment of taurine at 1% in drinking water prevented high-fat diet-induced increase in cholesterol levels of serum and liver. The decrease in serum cholesterol by taurine was due to decrease in non-HDL cholesterols. A similar tendency was noted in serum and liver cholesterol levels of hamsters fed a normal diet. In hamsters fed a high-fat diet, taurine prevented elevation in hepatic activity of acyl-CoA:cholesterol acyltransferase (ACAT) and increased the activity of cholesterol 7alpha-hydroxylase. Taurine also increased cholesterol 7alpha-hydroxylase activity in hamsters fed normal chow. Studies on liver membranes revealed that taurine increased 125I-labeled LDL binding by 52% and 58% in hamsters fed either a normal chow or high-fat diet, respectively. Furthermore, LDL kinetic analysis showed that taurine intake resulted in significant faster plasma LDL fractional catabolic rates (FCR). These results suggest that taurine elevates hepatic LDL receptor and thereby decreases serum cholesterol levels, an event which may be the result of hepatic cholesterol depletion as a consequence of increased bile acid synthesis via enhancement of cholesterol 7alpha-hydroxylase activity. Thus, up-regulation of the LDL receptor and subsequent increase in receptor- mediated LDL turnover may be a key event in the cholesterol-lowering effects of taurine in hamsters.     

Analysis of the Relationship between Cellular Thymidine Kinase Activity and Virulence of Thymidine Kinase-Negative Herpes Simplex Virus Types 1 and 2

The virulence of thymidine kinase-negative herpes simplex virus type 1 (HSV-1; VRTK^? strain) and type 2 (HSV-2; UWTK^? strain) was studied in comparison with that of their parental strains (VR-3 and UW-268, respectively) in an encephalitis model of adult (4-week-old) and newborn (3-day-old) mice. Viral thymidine kinase (TK) activity was essential for the maximum expression of virulence of HSV-1, because the 50% lethal dose (LD₅₀) of VRTK^? was 60 times higher than that of VR-3 in the brains of newborn mice expressing high levels of cellular TK activity. However, the UWTK^? strain showed the same virulence as the parental strain in newborn mice, despite the lack virulence in adults, suggesting that replication of the UWTK^? strain was completely supported by cellular TK activity. This difference in the role of viral and cellular TKs for virus growth between HSV-1 and HSV-2 was confirmed with the one-step growth of virus strains in L-M and L-M(TK^?) cells.     

Expression and characterization of Rab38, a new member of the Rab small G protein family

Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [alpha-32P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.     

Occurrence of the primary cell wall polysaccharide rhamnogalacturonan II in pteridophytes, lycophytes, and bryophytes. Implications for the evolution of vascular plants

Borate ester cross-linking of the cell wall pectic polysaccharide rhamnogalacturonan II (RG-II) is required for the growth and development of angiosperms and gymnosperms. Here, we report that the amounts of borate cross-linked RG-II present in the sporophyte primary walls of members of the most primitive extant vascular plant groups (Lycopsida, Filicopsida, Equisetopsida, and Psilopsida) are comparable with the amounts of RG-II in the primary walls of angiosperms. By contrast, the gametophyte generation of members of the avascular bryophytes (Bryopsida, Hepaticopsida, and Anthocerotopsida) have primary walls that contain small amounts (approximately 1% of the amounts of RG-II present in angiosperm walls) of an RG-II-like polysaccharide. The glycosyl sequence of RG-II is conserved in vascular plants, but these RG-IIs are not identical because the non-reducing L-rhamnosyl residue present on the aceric acid-containing side chain of RG-II of all previously studied plants is replaced by a 3-O-methyl rhamnosyl residue in the RG-IIs isolated from Lycopodium tristachyum, Ceratopteris thalictroides, Platycerium bifurcatum, and Psilotum nudum. Our data indicate that the amount of RG-II incorporated into the walls of plants increased during the evolution of vascular plants from their bryophyte-like ancestors. Thus, the acquisition of a boron-dependent growth habit may be correlated with the ability of vascular plants to maintain upright growth and to form lignified secondary walls. The conserved structures of pteridophyte, lycophyte, and angiosperm RG-IIs suggests that the genes and proteins responsible for the biosynthesis of this polysaccharide appeared early in land plant evolution and that RG-II has a fundamental role in wall structure.