Immunogenicity and protective effect against oral colonization by Streptococcus mutans of synthetic peptides of a streptococcal surface protein antigen

Streptococcus mutans is known to be a major causative organism of human dental caries. A surface protein Ag with a molecular mass of 190 kDa of S. mutans (PAc) is receiving attention as an anticaries vaccine. We have recently determined the complete nucleotide sequence of the gene for PAc. In this study, four peptides were synthesized on the basis of amino acid sequence of PAc. Among these peptides, PAc(301-319) corresponding to the alanine-rich repeating amino acid region was the most strongly bound by polyclonal murine anti-rPAc antibodies. The peptide partially inhibited the binding of polyclonal anti-rPAc antibodies to rPAc. The peptide induced the proliferation of T cells from BALB/c mice immunized with rPAc. Subcutaneous immunization with PAc(301-319) or rPAc emulsified in CFA/IFA induced high serum IgG responses to rPAc and PAc(301-319). In addition, serum IgG responses to a surface protein Ag with a molecular mass of 210 kDa of Streptococcus sobrinus were elicited in mice immunized by s.c. injection with PAc(301-319) or rPAc. Intranasal immunization with PAc(301-319) coupled to cholera toxin B subunit (CTB) or with rPAc and free CTB induced high serum IgG responses to rPAc. The immunization with PAc(301-319) coupled to CTB or rPAc and free CTB suppressed the colonization of murine teeth by S. mutans. These results suggest that intranasal immunization with the peptide or rPAc may be effective for the prevention of dental caries.     

Interactions of functionalized polymeric liposomes having a porphinato-iron complex with some biological cells and components in vitro

The hemocompatibility of functionalized polymeric liposome particles (diameter: 20-32 nm), which have a synthetic porphinato-iron complex in their polymerized bilayers and can carry oxygen, was studied in vitro. The ultramicroparticles did not induce hemolysis, platelet aggregation and plasma coagulation directly and were stable against hydrolysis by phospholipases A2 and D.     

Development and characterisation of microsatellite loci in Farfugium japonicum (Asteraceae)

Eight microsatellite loci for the perennial herb Farfugium japonicum, including the rheophytic variety luchuense endemic to riparian areas of the Ryukyu Islands, Japan, were isolated and characterised. The number of alleles ranged from 5 to 14. The expected (H _E) and observed (H _O) heterozygosities were 0.344–0.885 and 0.121–0.754, respectively, from 69 individuals in one population. Six loci exhibited significantly fewer heterozygotes than expected under Hardy–Weinberg equilibrium (P < 0.05). The primers amplifying microsatellite sequences in F. japonicum may provide a population genetics tool useful in the establishment of a conservation strategy.     

Absence of ganglioside GM1 in astroglial cells from newborn rat brain

An immunohistochemical method utilizing anti-ganglioside GM1 antiserum combined with the peroxidase-antiperoxidase technique was applied to a mixed cell population in primary cultures of newborn rat brain. Ganglioside GM1 was demonstrated to be present in neurons and oligodendroglia, but was absent in astroglia. This demonstration was confirmed using a newly developed biotinylated choleragen-avidin-peroxidase procedure. Primary cultures from newborn rat brain cells that had been subjected to a single treatment with trypsin (first passage) and then cultured for 14 days were predominately (95%) composed of astrocytes that stained positively for glial fibrillary acidic protein but were negative for GM1 ganglioside. This preparation contained only 0.34 nmol ganglioside NeuNAc per mg protein compared to 23.9 nmol gangliosidic NeuNAc/mg protein for a five day culture of newborn rat brain mixed cell culture that had not been subjected to passage. Prolongation of culture time from 5 to 21 days in the latter preparation reduced the ganglioside NeuNAc content to 4.9 nmol gangliosidic NeuNAc/mg protein as the proportion of astrocytes in the culture increased. Ganglioside GM1 could not be detected by TLC analysis of the lipid extract obtained from the “pure” astrocyte culture, although small amounts of GM3 and some polysialogangliosides were detected. About half of the label incorporated upon 24 h incubation of astrocytes in the presence of $\text{N-[}{}^3\text{H]acetylmannosammine}$ appeared in ganglioside GM3. It is concluded that astrocytes in mixed cell primary cultures from newborn rat brain, as well as astrocytes in astroglial preparations derived from such cultures, do not contain ganglioside GM1.     

Effect of low-temperature bovine ovary storage on the maturation rate and developmental potential of follicular oocytes after in vitro fertilization, parthenogenetic activation, or somatic cell nucleus transfer

The present study examined the competence of oocytes from bovine ovaries stored at low temperatures for at least 1 day, which is the necessary time period to complete inspection for bovine spongiform encephalopathy. Storage of ovaries at 10 degrees C for 24 h did not affect oocyte maturation (68% versus 68%) or the potential of oocytes to develop into day 8 blastocysts after in vitro fertilization (25% versus 27%), parthenogenetic activation (19% versus 25%), or somatic cell nucleus transfer (27% versus 32%) compared with controls. In vitro-fertilized and parthenogenetic oocytes from ovaries stored at 10 degrees C for 48 h had a significantly decreased maturation rate and developmental potential, but nucleus-transferred oocytes that received cultured cumulus cells did not (27% versus 32%). Thus, bovine ovaries can be stored at 10 degrees C for at least 24 h without decreasing oocyte maturation competence or the developmental potential of in vitro-fertilized, parthenogenetically activated, and somatic cell nucleus-transferred oocytes, at least to the blastocyst stage. The present study provides valuable information with regard to removing bovine ovaries from abattoirs after testing for bovine spongiform encephalopathy.     

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.     

Efficient Protein Expression in <Emphasis Type="Italic">Bombyx mori</Emphasis> Larvae of the Strain d17 Highly Sensitive to <Emphasis Type="Italic">B. mori</Emphasis> Nucleopolyhedrovirus

The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a “factory” for large-scale expression using the BmNPV bacmid system.     

Expression, purification, and crystallization of endopolygalacturonase from a pathogenic fungus, Stereum purpureum, in Escherichia coli

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.