In vitro gene transfection using dendritic poly(L-lysine)

Monodispersed dendritic poly(L-lysine)s (DPKs) of several generations were synthesized, and their characteristics as a gene transfection reagent were then investigated. The agarose gel shift and ethidium bromide titration assay proved that the DPKs of the third generation and higher could form a complex with a plasmid DNA, and the degree of compaction of the DNA was increased by the increasing number of the generation. The DPKs of the fifth and sixth generation, which have 64 and 128 amine groups on the surface of the molecule, respectively, showed efficient gene transfection ability into several cultivated cell lines without significant cytotoxity. In addition, the transfection efficiency of the DPK of the sixth generation was not seriously reduced even if serum was added at 50% of the final concentration into the transfection medium. Because we can strictly synthesize various DPK derivatives, which have several types of branch units, terminal cationic groups, and so on, they are expected to be a good object of study regarding the basic information on the detailed mechanism of gene transfection into cells. We also expect to be able to easily construct DPK-based functional gene carriers, e.g., DPKs modified by ligands such as a sugar chain, which can enable advanced gene delivery in vivo.     

Natural resistance to Clover yellow vein virus in beans controlled by a single recessive locus

We characterized the resistance of the common bean cv. Jolanda to Clover yellow vein virus no. 30 (ClYVV). After inoculation, the virus was detected in neither inoculated nor upper leaves, suggesting that the resistance operates at either the viral replication or cell-to-cell movement level. To analyze the mechanism of resistance, we developed a green fluorescent protein (GFP)-tagged ClYVV, and monitored GFP fluorescence at sites of infection on ClYVV-inoculated leaves. No GFP fluorescence was detected in Jolanda, whereas its expression in single cells and spread on inoculated leaves were observed clearly in susceptible cultivars. ClYVV-introduced Jolanda cells were found to be still viable; therefore, it is unlikely that the restriction of multiplication was due to rapid cell death. Genetic analysis indicated that a single recessive locus controlled the resistant phenotype of Jolanda. We designated this locus desc (determinant of susceptibility to ClYVV). Meanwhile, a spontaneous mutant virus that overcomes the resistance (ClYVV-Br) was isolated. Inoculation assays using chimeric viruses suggested that a viral genome-linked protein (VPg) might be the avirulence determinant. The resistance mechanism may be associated with the role of VPg in the viral infection cycle.     

Detection of 1270 nm emission from singlet oxygen and photocytotoxic property of sugar-pendant 60 fullerenes

Sugar-pendant [60] fullerene derivatives have been prepared from carbohydrate-linked azides 1a-e. Both monosugar (4a-e) and bissugar derivatives (5a-e) produce singlet oxygen ((1)O(2)) under laser irradiation (355 nm) proved by the direct observation of (1)O(2) emission at 1270 nm. Monosugar derivatives exhibit photocytotoxicity varying by the attached sugar molecule.     

Acid hydrolase activity of cultured bovine oligodendrocytes

We measured the activity of several acid hydrolases of cultured oligodendrocytes prepared from adult bovine brain white matter to clarify the biochemical basis of bovine oligodendrocytes in vitro. Lysosomal enzyme activities were assayed by using 4-methylumbelliferyl glycosides as substrates. Lysosomal enzyme activities became higher at 8–11 days in vitro (DIV) than 4 DIV. The enrichment in acid hydrolase specific activities in oligodendrocytes may be associated with lysosomal origin of myelin-like membranes.     

Morphologic differentiation of HL-60 cells is associated with appearance of RPTPbeta and induction of Helicobacter pylori VacA sensitivity

Phorbol 12-myristate 13-acetate (PMA) induces differentiation of human leukemic HL-60 cells into cells with macrophage-like characteristics and enhances the susceptibility of HL-60 cells to the Helicobacter pylori VacA toxin (de Bernard, M., Moschioni., M., Papini, E., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998) FEBS Lett. 436, 218-222). We examined the mechanism by which HL-60 cells acquire sensitivity to VacA, in particular, looking for expression of RPTPbeta, a VacA-binding protein postulated to be the VacA receptor (Yahiro, K., Niidome, T., Kimura, M., Hatakeyama, T., Aoyagi, H., Kurazono, H., Imagawa, K., Wada, A., Moss, J., and Hirayama, T. (1999) J. Biol. Chem. 274, 36693-36699). PMA induced expression of RPTPbeta mRNA and protein as determined by RNase protection assay and indirect immunofluorescence studies, respectively. Vitamin D(3) and interferon-gamma, which stimulate differentiation of HL-60 cells into monocyte-like cells, also induced VacA sensitivity and expression of RPTPbeta mRNA, whereas 1. 2% Me(2)SO and retinoic acid, which stimulated the maturation of HL-60 into granulocyte-like cells, did not. RPTPbeta antisense oligonucleotide inhibited induction of VacA sensitivity and expression of RPTPbeta. Double immunostaining studies also indicated that newly expressed RPTPbeta colocalized with VacA in PMA-treated HL-60 cells. In agreement with these data, BHK-21 cells, which are insensitive to VacA, when transfected with the RPTPbeta cDNA, acquired VacA sensitivity. All data are consistent with the conclusion that acquisition of VacA sensitivity by PMA-treated HL-60 cells results from induction of RPTPbeta, a protein that functions as the VacA receptor.     

Clinical effects of elastic bandage on neurogenic orthostatic hypotension

Neurogenic orthostatic hypotension (OH) often causes troublesome symptoms such as dizziness, syncope and falling, interfering active daily life or various therapies in rehabilitation. Nonpharmacologic measures for treating patients with OH include wearing elastic leotard, head-up tilting at night, etc. Elastic garment or antigravity suits is certainly effective, but it may be uncomfortable and not practical. Although elastic bandage (EB) bound on the lower limbs has been thought to be useful, there is few clinical report about its beneficial evidence. We investigated short-term clinical effects of commercially available EB on OH, and estimated the mechanism of its effectiveness by measuring some blood pressure-related humoral variables in neurodegenerative patients with OH.     

Detection of new anti-neutral glycosphingolipids antibodies and their effects on Trk neurotrophin receptors

We screened sera from patients with various neurological disorders for the presence of anti-neutral glycosphingolipids antibodies and only found them in sera from relapsing polychondritis with limbic encephalitis patients. Neutral glycosphingolipids are resident in membrane lipid rafts where high affinity nerve growth factor (NGF) receptor, Trk is co-localized. Therefore, we examined whether these antibodies influence the action of NGF in NGF-responsive cells. The results strongly suggest that these antibodies enhance NGF-induced Trk autophosphorylation and neurite outgrowth as well as neurofilament M expression. These data strongly indicate that these anti-neutral glycosphingolipids antibodies have a functional impact on NGF-Trk-mediated intracellular signal transduction pathway.     

Lymphopenia induced by a novel selective S1P1 antagonist structurally unrelated to S1P

Sphingosine 1-phosphate (S1P) regulates lymphocyte trafficking via type-1 S1P receptor (S1P₁) and participates in many pathological conditions. We developed a novel type S1P₁-selective antagonist, TASP0251078, which is structurally unrelated to S1P. This competitive antagonist inhibited binding of S1P to S1P₁ resulting in reduced signaling downstream of S1P₁, including GTPγS-binding and cAMP formation. TASP0251078 also inhibited S1P-induced cellular responses such as chemotaxis and receptor-internalization. Furthermore, when administered in vivo, TASP0251078 induced lymphopenia in blood, which is different from previously reported effects of other S1P₁-antagonists. In a mouse contact hypersensitivity model, TASP0251078 effectively suppressed ear swelling, leukocyte infiltration, and hyperplasia. These findings provide the chemical evidence that S1P₁ antagonism is responsible for lymphocyte sequestration from the blood, and suggest that the effect of S1P₁ agonists on lymphocyte sequestration results from their functional antagonism.     

Involvement of CD14 in the inhibitory effects of dimethyl-alpha-cyclodextrin on lipopolysaccharide signaling in macrophages

The potential use of alpha-cyclodextrin and its hydrophilic alpha-cyclodextrin derivatives (alpha-CyDs) as antagonists against lipopolysaccharide (LPS), which stimulates the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production as well as nuclear factor-kappaB (NF-kappaB) activation in macrophages was examined. Of three alpha-CyDs used in the present study, 2,6-di-O-methyl-alpha-CyD (DM-alpha-CyD) had greater inhibitory activity than did the other CyDs against NO and TNF-alpha production through an impairment of gene expression in macrophage cell lines and primary macrophages stimulated with LPS and lipid A in a concentration-dependent manner. Concomitantly, DM-alpha-CyD inhibited NF-kappaB translocation into nucleus. These inhibitory effects of DM-alpha-CyD could be attributed to the release of CD14 from lipid rafts caused by an efflux of phospholipids, but not cholesterol. These results suggest that DM-alpha-CyD may have promise as a potent and unique antagonist for excess activation of macrophages stimulated with LPS.