Application of retrovirus-mediated expression cloning for receptor screening of a parasite

Retrovirus-mediated expression cloning has been applied in both virology and cell biology. Although there is some difficulty in applying this technique to screening for a receptor recognized by an intracellular parasite, we modified the conventional method to identify a putative receptor for the Plasmodium falciparum BAEBL protein. We show that this method is effective in screening for a parasite receptor.     

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.     

Antibody against single-stranded DNA detects both programmed cell death and drug-induced apoptosis

Cyclophosphamide induced fragmented nuclei in mouse thymic epithelial cells. Agarose gel electrophoresis showed the fragmentation of the DNA extracted from mouse thymus exposed to cyclophosphamide. The cell death induced by cyclophosphamide was considered to be apoptotic. Polyclonal antibody against single-stranded DNA was used immunohistochemically to detect apoptotic cell death in thymic epithelial cells. This antibody also detected programmed cell death in the interdigital necrotic zone of the mouse limb plate on day 14 of gestation, and in the ganglion of the trigeminal nerve on day 13 of gestation. These results show that the antibody specific for single-stranded DNA detected both drug-induced apoptosis and programmed cell death during embryogenesis.     

Red cell NADH diaphorase variants in Japanese

Human red cell NADH diaphorase isozyme patterns were examined in 5,046 healthy adult Japanese by starch gel electrophoresis. Twenty had Dia 2-1 and 3 had Dia 4-1 phenotypes. The incidence of Dia variants in patients with mental retardation, cerebral palsy, epilepsy and Down's syndrome was also examined and compared with that of healthy people. It was noticed that thin-layer isoelectric focusing on polyacrylamide gel was very useful for discriminating variant bands from 'aging bands'.     

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation

Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPβ, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.     

%Hb A2, %Hb F, %G gamma values and the haplotypes in the beta-globin gene cluster in Japanese adults with elevated Hb F.

The percentages of minor adult hemoglobin (%Hb A2) in hemolysates and G gamma-globin chain (%G gamma) in fetal Hb (Hb F) of 15 individuals with elevated Hb F levels (2.0-11%) among 11,000 healthy Japanese adults were examined. Most of them might be carriers for the determinants of hereditary persistence of fetal hemoglobin. Subjects with less than 1.3% Hb A2, some of whom might be also carriers for delta-thalassemia determinants, had high G gamma values (54-70%). Those homozygous for a subhaplotype [+-----] 5' to the delta-globin gene had low to mid G gamma values (7-49%), while those homozygous for [-++-++] possessed high G gamma values (60-85%), but varied Hb F values (3.1-11%). Those heterozygous for the presence of the XmnI site 5' to (-158 bp to the cap site of) the G gamma-globin gene had mid to high G gamma values (53-65%). Factors for the high or low G gamma-globin gene expression in the Japanese adult with elevated Hb F level should be highly associated with a subhaplotype [-++-++] or [+-----], respectively.     

Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides

Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC₅₀ 7.0 μM) among the tested compounds.     

Molecular analyses of Toxoplasma gondii calmodulin-like domain protein kinase isoform 3

Ca²⁺ signaling is thought to play an important role in Toxoplasma gondii motility, including invasion of and egress from host cells. Recently, it has been reported that phosphorylation of the glideosome apparatus components of T. gondii occurs during invasion. To elucidate the role of T. gondii calmodulin-like domain protein kinase in the signaling pathway that bridges Ca²⁺ stimulation and motility, we characterized T. gondii calmodulin-like domain protein kinase isoform 3 (TgCDPKif3). TgCDPKif3 is homologous to Plasmodium falciparum calcium-dependent protein kinase 1, which has been reported to phosphorylate P. falciparum glideosome components. TgCDPKif3 was purified as a fusion protein that was labeled with [γ-³²P]ATP, and the label was subsequently removed by phosphatase treatment. Phosphorylation was eliminated when the putative catalytic lysine residue of TgCDPKif3 was replaced with alanine. TgCDPKif3 phosphorylated Histone II_AS as a representative substrate in a Ca²⁺-dependent manner at a high Ca²⁺ concentration. TgCDPKif3 was localized to the apical ends of tachyzoites. TgCDPKif3 showed the translocation between intra- and extracellular tachyzoites. TgCDPKif3 could phosphorylate T. gondii aldolase 1 (TgALD1) in vitro. The interaction between TgCDPKif3 and TgALD1 was confirmed by the co-immunoprecipitation assay in mammal cells. We suggested that TgCDPKif3 could participate in the motility of T. gondii through the phosphorylation of glideosome complex member.     

A Single-Amino-Acid Substitution in Herpes Simplex Virus 1 Envelope Glycoprotein B at a Site Required for Binding to the Paired Immunoglobulin-Like Type 2 Receptor α (PILRα) Abrogates PILRα-Dependent Viral Entry and Reduces Pathogenesis

Paired immunoglobulin-like type 2 receptor α (PILRα) is a herpes simplex virus 1 (HSV-1) entry receptor that associates with O-glycans on HSV-1 envelope glycoprotein B (gB). Two threonine residues (Thr-53 and Thr-480) in gB, which are required for the addition of the principal gB O-glycans, are essential for binding to soluble PILRα. However, the role of the two threonines in PILRα-dependent viral entry remains to be elucidated. Therefore, we constructed a recombinant HSV-1 carrying an alanine replacement of gB Thr-53 alone (gB-T53A) or of both gB Thr-53 and Thr-480 (gB-T53/480A) and demonstrated that these mutations abrogated viral entry in CHO cells expressing PILRα. In contrast, the mutations had no effect on viral entry in CHO cells expressing known host cell receptors for HSV-1 gD, viral entry in HL60 cells expressing myelin-associated glycoprotein (MAG) (another HSV-1 gB receptor), viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. These results support the hypothesis that gB Thr-53 and Thr-480 as well as gB O-glycosylation, probably at these sites, are critical for PILRα-dependent viral entry. Interestingly, following corneal inoculation in mice, the gB-T53A and gB-T53/480A mutations significantly reduced viral replication in the cornea, the development of herpes stroma keratitis, and neuroinvasiveness. The abilities of HSV-1 to enter cells in a PILRα-dependent manner and to acquire specific carbohydrates on gB are therefore linked to an increase in viral replication and virulence in the experimental murine model.Herpes simplex virus 1 (HSV-1) entry into host cells depends on interactions between cell surface receptors and HSV-1 virion envelope glycoproteins (39). Five of the 12 HSV-1 envelope glycoproteins that have been identified thus far (i.e., glycoprotein B [gB], gC, gD, gH, and gL) have roles in viral entry (39). Both gB and gC mediate virion attachment by interacting with cell surface glycosaminoglycan, primarily heparan sulfate (16, 17). Although not essential for entry, this step provides stable interactions between the virion and the cell that favor the next steps (39). These steps include gD binding to one of its identified receptors, i.e., herpesvirus entry mediator (HVEM), nectin-1, and specific sites on heparan sulfate 3-O-sulfated heparan sulfate (3-O-S-HS) generated by certain 3-O-sulfotransferases (3-O-STs) (14, 28, 38, 51). Subsequent fusion between the virion envelope and host cell membrane, which requires the cooperative function of gB, heterodimer gH/gL, gD, and a gD receptor, then produces nucleocapsid penetration into the cell (31, 46).In addition to the interaction of gD with a gD receptor, gB binding to a cellular receptor other than heparan sulfate has been suggested to mediate viral entry, based on the observation that a soluble form of gB binds to heparan sulfate-deficient cells and blocks HSV-1 infection of some cell lines (3). Consistent with this observation, we have reported that paired immunoglobulin-like type 2 receptor α (PILRα) associates with gB and functions as an HSV-1 entry receptor (36). Viral entry via PILRα appears to be conserved among alphaherpesviruses, but there is a PILRα preference based on the observation that PILRα is able to mediate the entry of pseudorabies virus, a porcine alphaherpesvirus, but not of HSV-2 (1). Importantly, HSV-1 infection of human primary monocytes expressing both HVEM and PILRα was blocked by either an anti-PILRα or anti-HVEM antibody, suggesting that cellular receptors for both gD and gB are required for HSV-1 infection (36). However, CHO-K1 cells, which are resistant to HSV-1 infection, can become susceptible to HSV-1 entry and HSV-1-induced cell fusion after the overexpression of either a gD receptor, such as nectin-1, or PILRα (14, 36). It was thought that CHO-K1 cells express endogenously low levels of gB and gD receptors that allow the single overexpression of either a gB or gD receptor to support detectable levels of HSV-1 entry and HSV-1-induced cell fusion (36). More recently, myelin-associated glycoprotein (MAG), which has homology to PILRα, was also reported to serve as the gB receptor for HSV-1 and varicella-zoster virus (40). However, the importance of PILRα- or MAG-dependent viral entry in HSV-1 infection and pathogenesis in vivo remains to be elucidated.PILRα is one of the paired receptor families, in which one receptor has inhibitory functions and the other mediates activation functions, and is expressed mainly in immune system cells (13, 29). In addition, PILRα was previously reported to be expressed in certain types of cells in neural tissues (36). We previously identified one of the PILRα ligands as CD99 (37). Interestingly, PILRα recognition of CD99 is dependent on the addition of sialylated O-linked sugar chains at particular CD99 threonines (50). Similarly, we recently demonstrated that a specific sialylated O-glycan(s) on gB is critical for PILRα binding, based on observations that neuraminidase, which removes sialic acid, and benzyl-α-GalNAc treatment, which blocks O-glycan synthesis, inhibited gB binding to a soluble PILRα (49). More importantly, one (Thr-53) or both (Thr-53 and Thr-480) putative O-glycosylation sites identified by bioinformatics analysis are required for the binding of gB to soluble PILRα, and the replacement of both Thr-53 and Thr-480 with alanine significantly inhibited the addition of O-glycans to gB (49). These observations suggest that Thr-53 and Thr-480 in gB are O-glycosylated, and these sites, and probably the addition of specific carbohydrates to them, are required for the interaction of gB with PILRα. However, it remains uncertain whether gB Thr-53 and Thr-480, and probably the gB O-glycosylation of these sites, are required for PILRα-dependent viral entry in natural infections.In the present study, we have shown that the alanine replacement of gB Thr-53 (gB-T53A) alone or of both gB Thr-53 and Thr-480 (gB-T53/480A) significantly inhibited cell-cell fusion in CHO cells expressing PILRα, gB, gD, gH, and gL, whereas the mutations had no effect on cell-cell fusion in CHO cells expressing nectin-1, gB, gD, gH, and gL. Furthermore, we constructed recombinant HSV-1 carrying the gB-T53A and gB-T53/480A mutations and found that these mutations abrogated PILRα-dependent viral entry but had no effect on viral entry via known receptors for HSV-1 gD and MAG, viral attachment to heparan sulfate, and viral replication in PILRα-negative cells. We also tested these recombinant viruses in mice and present data showing that the mutations in gB significantly reduced viral replication, the development of herpes stromal keratitis (HSK), and neuroinvasiveness.