[Comparative study on the ability of adhesion and invasion of different fimA genetypes of Porphyromonas gingivalis to oral epithelial cells].

OBJECTIVE: To compare the ability of adhesion and invasion to epithelial cells by Porphyromonas gingivalis (Pg) strains with different fimA separated from Chinese. METHODS: Cultured method and antibiotic protection method were used to determine the adhesive and invasive ability of Pg with different fimA genetypes. The adhesion was observed by scanning electron microscope. RESULTS: All the strains adhered and invaded to KB cells, and the adhesion rate ranged from 0.523% to 37.125% and invasive rate from 0.017% to 3.750%.The adhesive and invasive ability among different fimA genotypes showed no significant difference (P > 0.05). CONCLUSIONS: There is no significant correlation between fimA genotype and ability in adhesion and invasion to KB cells.

Protein crystals in Adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis.

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489-492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors.

The influence of di-acetylation of the hydroxyl groups on the anti-tumor-proliferation activity of lutein and zeaxanthin.

Lutein and zeaxanthin have various activities such as anti-age-related macular degeneration, anticataract, anticancer and cardiovascular diseases risk lowering, etc.; however, few studies have been reported on the role of free hydroxyl groups in the antitumor effects of lutein and zeaxanthin. The structure-activity relationship (SAR) of lutein and zeaxanthin di-acetylation derivatives has been investigated. The lutein and zeaxanthin were purified from corn protein residues and structurally modified by di-acetylation, which was characterized with UV/visible and HPLC/MS spectroscopy. The anti-proliferative effects of di-acetylated lutein or zeaxanthin on the human mouth epithelial cancer line KB cell were compared with controls of their original counterparts. Results showed that the anti-tumor activities of the di-acetylation of lutein and di-acetylation of zeaxanthin decreased significantly (p<0.05) at 50 micromol/L. Also the related IC50 dose was increased after di-acetylation. It was suggested that the hydroxyl groups contribute to the anti-tumor activity of lutein and zeaxanthin.

Atomic force microscopy study of early morphological changes during apoptosis.

Apoptosis is defined by a distinct set of morphological changes observed during cell death including loss of focal adhesions, the formation of cell membrane buds or blebs, and a decrease in total cell volume. Recent studies suggest that these dramatic morphological changes, particularly apoptotic volume decrease (AVD), are an early prerequisite to apoptosis and precede key biochemical time-points. Here we use atomic force microscopy to observe early stage AVD of KB cells undergoing staurosporine-induced apoptosis. After a 3-h exposure to 1 microM staurosporine, a 32% decrease in total cell height and a 50% loss of total cell volume is observed accompanied by only a 15% change in cell diameter. The observed AVD precedes key biochemical hallmarks of apoptosis such as loss of mitochondrial membrane potential, phosphatidyl serine translocation, nuclear fragmentation, and measurable caspase-3 activity. This suggests that morphological volume changes occur very early in the induction of apoptosis.

Evaluation of the in vitro biocompatibility of various elastomers.

A previous study highlighted the superior shock absorption of silicone rubbers compared to other elastomers. We evaluated and compared the in vitro biocompatibility of silicone-based rubbers and propose them as an alternative to conventional products. We used the MTT colorimetric test to assess cell viability and flow cytometry to evaluate cell proliferation. Tests were conducted at 24 and 72 h. Changes in cell morphology were evaluated by scanning electron microscopy. Positive (polyurethane) and negative (polystyrene) toxicity controls were included. The number of viable cells was significantly higher on polystyrene than on polyurethane. A decrease in the total number of cells from 24 to 72 h compared to the negative control was correlated with a lower percentage of S-phase cells. The differences in cell viability noted between the samples and the polystyrene control mainly resulted from an initial lack of adhesion, which was confirmed by scanning electron microscopy. The biocompatibility of the three silicone rubbers was comparable to the best of the three products currently being used. These results, combined with those of the previous study, indicate that silicone rubber could be considered for the manufacture of mouth guards.

A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.

Isogenic mutants of Porphyromonas gingivalis which differ in the expression of fimbriae were used to examine the contribution of fimbriae in invasion of a human oral epithelial cell line (KB). At a multiplicity of infection of 100, the wild-type P. gingivalis strains 33277, 381, and A7436 exhibited adherence efficiencies of 5.5, 0.11, and 5.0%, respectively, and invasion efficiencies of 0.15, 0.03, and 0.10%, respectively. However, adherence to and invasion of KB cells was not detected with the P. gingivalis fimA mutants, DPG3 and MPG1. Adherence of P. gingivalis wild-type strains to KB cells was completely inhibited by the addition of hyperimmune sera raised to the major fimbriae. Examination by electron microscopy of invasion of epithelial cells by the P. gingivalis wild-type strain 381 revealed microvillus-like extensions around adherent bacteria; this was not observed with P. gingivalis fim mutants. Taken together, these results indicate that the P. gingivalis major fimbriae are required for adherence to and invasion of oral epithelial cells.

Retinoic acid-induced alteration of cell surface topography and other changes in an oral carcinoma cell line in culture.

Nature of antiproliferative action of retinoic acid (RA) on KB cells was studied by using monolayer and agar culture techniques. RA-treated cultures showed increased requirement of serum for their growth. Growth of colonies in agar culture was significantly retarded when cells were treated with 40 mumol RA. RA-induced growth inhibitions in both monolayer and agar cultures were independent of cell seeding densities. Cortisone and hydrocortisone showed no reversal of the inhibitory effects induced by RA on KB cells. Scanning electron microscopy study revealed a significant alteration in cell surface topography of RA-treated cells in monolayer culture. The results demonstrate that RA has a potential of reversing some of the properties which are associated with transformed state of oral carcinoma cells.

Estudo histoquímico comparativo da matriz extracelular de tumores primários e metastáticos em ratos nude xenotransplantados com células KB / Histochemical comparative study of primary and metastatic tumors in nude rates xenotransplanted with KB cells

Estudou-se comparativamente, através de métodos histoquímicos, a expressäo dos componentes da matriz extracelular de tumores primitivos e metastáticos em ratos nude, xenotransplantados com células KB. Em ambas as neoplasias observou-se uma variabilidade tanto qualitativa como quantitativa dos componentes matriciais, coexistência de diferentes tipos de fibras, pouca representatividade de fibras elásticas de glicosaminoglicanas ácidas e sulfatadas e de polissacarídeos neutros, além da ausência de membrana basal

Endocytosis of folate-protein conjugates: ultrastructural localization in KB cells.

It has been demonstrated that proteins covalently conjugated to folic acid may be taken up by cells via endocytosis after binding to a folate binding protein (FBP) in the cell membrane. The proteins taken up in this manner remain catalytically active and they may modify physiological processes occurring in the cytosol. Confocal fluorescence microscopy of KB cells incubated with FITC-bovine serum albumin-folic acid conjugates showed that after uptake, the conjugates resided in large vesicular structures. The purpose of the present study was to determine the subcellular localization of protein-folic acid conjugates in KB cells using folic acid-bovine serum albumin-colloidal gold (F-BSA-CG) as a tracer. F-BSA-CG conjugates were taken up via uncoated pits or caveolae, and resided primarily in multivesicular bodies (MVBs) and other tubular endosomes at early time points (15-60 min). At later time points (6 hours), conjugates were still contained in MVBs but some were also found in secondary lysosomes or free in the cytoplasm. Coincubation of KB cells with transferrin-colloidal gold (TF-CG) and F-BSA-CG resulted in colocalization of TF-CG and F-BSA-CG within endosomal elements at times later than 15 minutes, indicating that the caveolae-mediated F-BSA-CG endocytic pathway converged with a pathway utilized by clathrin-coated pits.

Stable transfectants of human MCF-7 breast cancer cells with increased levels of the human folate receptor exhibit an increased sensitivity to antifolates.

A major problem in cancer therapy is tumor drug resistance such as is found with antifolates (e.g., methotrexate [MTX]). We are specifically interested in the role of the human folate receptor (hFR) in MTX resistance. To investigate whether transfection of hFR results in increased MTX uptake and increased drug sensitivity, human mammary carcinoma (MCF-7) cells and Chinese hamster ovary cells (CHO) (cells which do not express detectable levels of hFR) were transfected with hFR cDNA. Stable human mammary carcinoma and Chinese hamster ovary transfectants expressing high levels of hFR were selected for further analysis. Transfected cells which express increased levels of hFR grow more rapidly than mock transfected or wild type cells in media containing physiologic concentrations of folates. The hFR expressed by these cells is sorted to the plasma membrane and is functional as determined by cell surface binding of a radiolabeled folic acid derivative and by internalization of [3H]methotrexate. The stable transfectants that express increased levels of hFR are also more sensitive to MTX in physiologic concentrations of folates. We conclude that increased expression of hFR by human mammary carcinoma and Chinese hamster ovary cells cultured under these conditions results in an enhanced growth rate, increased folic acid binding, and increased MTX uptake and cytotoxicity.

Subcellular distribution of spin-labeled bithiazoles and bleomycin in living KB cells: an ESR study.

Spin-labeled derivatives of bithiazole and bleomycin were studied with respect to their uptake and localization in KB cells in vivo. It was found that the amidification of the C-terminal carboxylic group of bleomycinic acid was essential for the penetration of the probes in the cells and that the subcellular localization depended on the number and spacing of positively charged groups.