Antimicrobial specificity and hemolytic activity of cyclized basic amphiphilic beta-structural model peptides and their interactions with phospholipid bilayers.

We synthesized a series of cyclic antiparallel beta-sheet model peptides with various ring sizes, which were designed on the basis of a cyclic beta-structural antibiotic, gramicidin S (GS); cyclo(Val-Orn-Leu-D-Phe-Pro)2, and investigated in terms of their antimicrobial activity and specificity against Gram-positive and Gram-negative bacteria and lytic activity for human erythrocytes. In our planning, in order to compare the peptides with GS, D-Phe-Pro sequence forming beta-turn in GS molecule remained unaltered and repeating sequences of alternately hydrophobic (Leu)-hydrophilic (Orn) residue were introduced into the beta-structural parts. CD study in acidic liposomes as well as leakage study of carboxyfluorescein encapsulated in phospholipid vesicles indicated that the peptides strongly interacted with lipid bilayers by taking an amphiphilic beta-structure. Antimicrobial study showed that although GS is active only against Gram-positive bacteria, the antimicrobial spectra of the model peptides transformed gradually to be active against Gram-negative ones and finally only against Gram-negative bacteria whose repeating sequences increased. It should be noted that the designed cyclic model peptides show antibacterial activity but accompany no hemolysis. This indicates that an appropriate hydrophobicity together with a proper orientation of hydrophilic (cationic) and hydrophobic groups in cyclic beta-structural molecules can hold antimicrobial activity against both types of bacteria without damaging eukaryotic cells.

Electrical detection of biomolecular interactions with metal-insulator-semiconductor diodes.

We report the label-free detection of DNA hybridization using a metal-insulator-semiconductor (MIS) diode or capacitor. Upon immobilization of single-stranded DNA on the gold gate of a MIS capacitor, the capacitance versus voltage characteristics show a significant shift in the direction of negative voltages as expected from the immobilization of negative charges on the gate. The hybridization with the complementary strand gives rise to a further significant shift in the same direction as before, which is consistent with the increase of negative charges on the gate brought about by the hybridization. Fluorescence studies indicate that the immobilization and hybridization of DNA can be electrostatically promoted by electric fields externally applied to the MIS capacitors. The MIS diode detection method is applicable to all biomolecular interactions that affect the surface dipole at the interface between the metal gate and the electrolyte and can be extended to other chemical and biochemical systems such as proteins and cells.

Contribution of IL-1 beta to the enhancement of Campylobacter rectus lipopolysaccharide-stimulated PGE2 production in old gingival fibroblasts in vitro.

Campylobacter rectus is associated with adult periodontitis. We previously reported that C. rectus lipopolysaccharide (LPS)-stimulated prostaglandin E2 (PGE2) production in old cells of human gingival fibroblasts (HGFs) is higher than that in young cells. The present study examined whether an enhancement of C. rectus LPS-stimulated interleukin (IL)-1 beta production in old HGFs contributed to the increased production of PGE2. LPS was prepared from C. rectus ATCC33238. HGFs were established from healthy gingiva in three patients, aged 10-12 years. Cellular aging in culture was determined with increasing doubling. The cultured cells were treated with LPS (0.01-10 micrograms/ml), and the amount of IL-1 beta in the medium was measured after a 24 h incubation. The LPS-stimulated IL-1 beta production in each old cell (corresponding to 57-67% of complete life-span) was increased (1.6-2.6 times) compared to that in the young cells (corresponding to 17-20% of the life-span). The IL-1 beta mRNA synthesis in the presence of LPS in the old cells was higher than that in the young cells. The enhancement of LPS-stimulated PGE2 production was inhibited by anti-IL-1 beta antibody and by IL-1 receptor antagonist. These findings suggest that the greater ability of old cells to produce PGE2 in response to C. rectus LPS is due to their greater level of IL-1 beta.

Effects of in vitro cellular aging on alkaline phosphatase, cathepsin activities and collagen secretion of human periodontal ligament derived cells.

It is believed that the degree of periodontal tissue breakdown and tooth loss increase with age. In periodontal tissues which are gingiva, periodontal ligament (PL), alveolar bone and tooth cementum, the PL which is soft connective tissue, lies between the tooth cementum and alveolar bone, having the primary function of tooth support, and maintaining the homeostasis of supporting tissues, as well as providing the healing process. We therefore investigated the effects of in vitro cellular aging on alkaline phosphatase (ALP), cathepsin activities and collagen secretion from human PL cells obtained from 18-23 year-old patients' teeth. ALP, cathepsin activities and collagen secretion may play important roles in the remodeling and maintaining of periodontal tissues. To investigate the life span of PL cells, the cells were sequentially subcultivated. The maximum population doubling level of the PL cells in the present experiment was 22-25 passages. Investigating some important biological activities of the PL cells at different passage levels (6-7, 30% of life span to 17-20, 75% of life span), ALP activity and collagen secretion were found to have significantly decreased while cathepsin B and L activities significantly increased with cellular aging. Since these biological activities in human PL cells tend to be more catabolic with increase in cellular aging, the increase in periodontal breakdown with age may be partly related to the catabolic changes of the PL cells themselves.

In vitro senescence enhances IL-6 production in human gingival fibroblasts induced by lipopolysaccharide from Campylobacter rectus.

The production of interleukin-6 (IL-6) in human gingival fibroblasts (Gin cells) is increased by lipopolysaccharide (LPS) from Campylobacter rectus (C. rectus), which is associated with adult periodontitis; however, the age-related changes in the susceptibility of Gin cells to C. rectus LPS remain unclear. We examined the influence of in vitro senescence on C. rectus LPS-stimulated IL-6 production in Gin cells. LPS was prepared from C. rectus ATCC 33238 using hot phenol-water. The Gin cells were established from healthy gingival tissue removed from three patients, aged 10-12 years. The cells were cultured until confluence then stimulated with LPS (0.01, 0.1, 1.0 and 10.0 micrograms/ml). Levels of IL-6 released in the medium were measured after incubation for 3, 6, 9, 12, and 24 h. In both young (5-6 population doublings) and senescent (17-20 population doublings) cells, LPS stimulated IL-6 production in a dose- and time-dependent manner. In response to 0.01-10.0 micrograms/ml of LPS, IL-6 production in the senescent cells was higher than that in the young cells. Using cells from each of the three donors, we found that this phenomenon of higher LPS-stimulated IL-6 production in senescent cells was reproducible. The greater capacity of the senescent cells to synthesize IL-6 in response to LPS was a higher production of mRNA for IL-6. This increase of IL-6 production induced by C. rectus LPS in senescent Gin cells could help to explain the increased susceptibility to periodontal diseases shown by aged individuals.

Polymerase chain reaction and an outer membrane protein gene probe for the detection of Porphyromonas gingivalis.

A sensitivity assay for Porphyromonas gingivalis based upon the polymerase chain reaction (PCR) was developed. A 426-bp sequence, including a DraI-HincII DNA fragment (278 bp) encoding the 40-kDa outer membrane protein of the P. gingivalis gene was amplified. PCR products were obtained from chromosomal DNAs of the P. gingivalis strains tested but not from those of other oral microorganisms. The lower limit of template DNA detection was 10 pg with 30 cycles and 100 fg with 40 cycles of PCR by agarose gel electrophoresis. The PCR products were hybridized with DraI-HincII DNA fragment internal to the PCR primers regions used. The lower limit of hybridization detection was 10 pg and 10 fg of template DNA with 30 and 40 cycles of PCR, respectively. These results demonstrated the simplicity, rapidity and specificity of the procedure, as well as the use of the DraI-HincII DNA fragment in the identification of P. gingivalis.

Glycylprolyl dipeptidylaminopeptidase from Bacteroides gingivalis.

Dipeptidyl aminopeptidase activity was found in the culture medium of Bacteroides gingivalis 381. The enzyme, hydrolyzing glycylprolyl-4-methylcoumaryl-7-amide, was purified 750-fold from culture medium by ammonium sulfate precipitation, Sephadex G-200 gel filtration, and DEAE Bio Gel A column chromatography. The molecular weight, determined by gel filtration, was approximately 160,000. The isoelectric point of the enzyme, estimated by isoelectric focusing using polyacrylamide disk gel electrophoresis, was about pH 6.2. The optimum pH of the enzyme was about 8.0, and the Km value was 0.05 mM. The enzyme activity was strongly inhibited by phenylmethylsulfonylfluoride and diisopropylfluorophosphate. The purified enzyme specifically cleaved glycylprolyl dipeptide from partially digested type I collagen.

Inhibition of prostaglandin E2 and interleukin 1-beta production by low-power laser irradiation in stretched human periodontal ligament cells.

It is well-known that orthodontic treatment usually causes some discomfort and pain to the patients. Recently, it has been reported that low-power laser irradiation is effective in reducing the pain accompanying tooth movement. However, the mechanism of such pain relief cannot be elucidated. Since high levels of prostaglandin (PG) E2 and interleukin (IL)-1 beta are found in the periodontal ligament (PDL) during tooth movement, and both factors are involved in the induction of pain, the effects of low-power laser irradiation on PGE2 and IL-1 beta production in stretched human PDL cells were studied in vitro. The PDL cells, derived from healthy premolars extracted for orthodontic treatment, were utilized for experiments. Cells were seeded in flexible-bottomed culture plates, and the bottom of each plate was elongated (18% increase) under vacuum at 6 cycles per min for 1, 3, or 5 days. The stretched cells were irradiated with a Ga-Al-As low-power diode laser (60 mW) once a day for 3, 6, or 10 min (from 10.8 to 36.0 J) for 1, 3, or 5 days. PGE2 and IL-1 beta levels in the medium were measured by radioimmunoassay. In response to mechanical stretching, human PDL cells showed a marked elevation in PGE2 production in a time-dependent manner. IL-1 beta production was also elevated, but this remained constant. The increase in PGE2 production was significantly inhibited by laser irradiation in a dose-dependent manner. The increase in IL-1 beta production was also significantly inhibited by laser irradiation, although the inhibition was only partial.(ABSTRACT TRUNCATED AT 250 WORDS)

Bactericidal activity of a monoclonal antibody against a recombinant 40-kDa outer membrane protein of Porphyromonas gingivalis.

BACKGROUND: We have cloned the gene for a 40-kDa outer membrane protein (40-kDa OMP) from Porphyromonas gingivalis 381. The recombinant (r)40-kDa OMP has become the subject of considerable interest because of its potential role in the development of a vaccine useful for passive immunization. To develop such a vaccine, it is essential to fully understand the functions of anti-r40-kDa OMP antibody in the host defense against P. gingivalis. To that end, we developed a panel of monoclonal antibodies by immunizing mice with purified r40-kDa OMP. The objective of this study was to determine the bactericidal activity on P. gingivalis by the IgG1 monoclonal antibody Pg-ompA2. METHODS: Bacterial growth measurement, a complement-mediated anti-P. gingivalis assay based on [3H]thymidine uptake, and a 14C-release assay were performed to test the bactericidal activity of Pg-ompA2 to P. gingivalis. RESULTS: In the presence of complement, Pg-ompA2 was lethal to P. gingivalis 381 as well as to the more virulent P. gingivalis strains, including ATCC 53977 and W83. Using component-deficient complement, we determined that Pg-ompA2 killed P. gingivalis by activating both the classical and alternative complement pathways. CONCLUSIONS: Pg-ompA2 has an in vitro complement-mediated bactericidal activity to P. gingivalis. Pg-ompA2 may contribute to the development of a local immunotherapy that can be applied in the gingival crevice of a patient with P. gingivalis-related periodontitis, or be a vaccine candidate.

Opsonophagocytic effect of antibody against recombinant conserved 40-kDa outer membrane protein of Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis is associated with the initiation and progression of adult periodontitis. The outer membrane proteins of the bacteria are potentially important targets for interaction with host defense systems. A 40-kDa outer membrane protein (40-kDa OMP) is conserved among many strains of P. gingivalis. We have cloned the gene for 40-kDa OMP from P. gingivalis 381 and produced a recombinant protein. For the development of recombinant 40-kDa OMP as a component of a vaccine for passive immunization, the elucidation of the roles of the anti-recombinant 40-kDa OMP antibody in the host defense against P. gingivalis is essential. The objective of this study was to determine the opsonic capacity of the antibody for phagocytosis by neutrophils which play a key role in the immune response to microbial infections. METHODS: To test the opsonic activity of a rabbit polyclonal antibody against r40-kDa OMP (r40-kDa OMP Ab) on human neutrophils to phagocytize P. gingivalis, we constructed a reproducible in vitro model of P. gingivalis-neutrophil interaction using the human promyelocytic cell line HL-60. RESULTS: We demonstrated that r40-kDa OMP Ab in the presence of human complement successfully opsonized [3H]-thymidine-labeled P. gingivalis as a target for phagocytosis by HL-60 cells differentiated with dimethyl sulfoxide. The phagocytized bacteria were then intracellularly killed and lysed, and the radioactive degradation debris egested into the culture medium. CONCLUSIONS: We conclude that antibody against r40-kDa OMP has opsonic activity on human neutrophil function for phagocytosis of P. gingivalis. Subgingival bacteria are coated in vivo with immunoglobulin and complement. When the antibody is specific for crevicular bacteria, immunological interactions can be expected in the crevice. Our observations suggest that the anti-recombinant 40-kDa OMP antibody in concert with the crevicular complement may prevent P. gingivalis colonization r40-kDa OMP may contribute to the development of a local immunotherapy when applied to the crevice of a patient with P. gingivalis-related periodontitis which relates to susceptibility for certain systemic diseases such as diabetes mellitus, cardiovascular disease, and preterm labor.

A sensitive method for detecting Porphyromonas gingivalis by polymerase chain reaction and its possible clinical application.

BACKGROUND: It is useful for the clinical diagnosis of periodontitis to monitor the colonization of periodontopathic bacteria in periodontal pockets. In this study, we attempted to establish and possibly identify the clinical application of a sensitive method to detect Porphyromonas gingivalis (P.g.), one of the putative periodontopathic bacteria related to chronic periodontitis. METHODS: Genomic DNA extracted from cultured P.g. 381 and clinically isolated subgingival plaque samples were used as a template of polymerase chain reaction (PCR). We designed primers to amplify the genomic DNA coding 40 kDa outer membrane protein (OMP), one of the unique proteins to all strains of P.g. The efficiency and specificity of amplification were evaluated by agarose gel electrophoresis and subsequent Southern hybridization with a digoxygenin-labeled oligonucleotide probe. RESULTS: Fewer than 100 P.g. bacterial cells in the specimen were reproducibly detected by PCR-hybridization assay. This PCR-hybridization assay was at least 100 times more sensitive than the conventional indirect immunofluorescence assay (IIF). Furthermore, the imaging analysis showed that there is a linear correlation between the strength of the signal and the cell number of P.g. from which the template DNA was extracted semiquantitatively. It is noteworthy that the PCR assay could also be applied to detect P.g. from clinical plaque samples and that it was approximately 100 times more sensitive than a conventional IIF assay. CONCLUSION: The PCR assay established in this study can be a powerful tool to detect P.g. in periodontal pockets and monitor the colonization and/or recolonization of P.g. at the very early phase.

Suppression of proliferation of a human B-cell leukaemic cell line derived from acute lymphoblastic leukaemia by soluble factor(s) from Campylobacter rectus.

Soluble sonic extracts of several strains were examined for their ability to alter proliferation of a cell line derived from acute lymphoblastic leukaemia (BALL-1). Extracts of all strains tested caused dose-dependent suppression of proliferation when assessed by DNA (tritiated thymidine incorporation), RNA (tritiated uridine incorporation) and protein (tritiated leucine incorporation) synthesis. There was no effect on the viability of BALL-1 as measured by either trypan-blue exclusion or extracellular release of the cytoplasmic enzyme lactate dehydrogenase. The suppressive factor(s) was separated in a well-defined peak by high-pressure liquid DEAE ion-exchange chromatography, which revealed a single active peak with a molecular mass of 48 kDa. Characterization of the peak indicated that the suppressive factor(s) was heat labile (activity destroyed at 80 degrees C) and sensitive to the proteolytic enzyme pronase P. The soluble suppressive factor(s) from Campylobacter rectus thus has protein-like properties and no cytotoxicity to a human B-cell leukaemic cell line.

Cloning of the gene for cell-surface protein antigen A from Streptococcus sobrinus (serotype d).

A gene library of Strep. sobrinus B13N (serotype d) chromosomal DNA was constructed in Escherichia coli, with the bacteriophage vector lambda L47.1. A recombinant phage, lambda MDSM49, containing a 15.5 kb DNA insert, directed the expression of a 210 kDa antigenic protein. The recombinant 210 kDa protein was shown by Western blot analysis to be identical with cell-surface protein antigen A (spaA) from a serotype g strain. However, the restriction patterns of a subclone plasmid, pMD51, from lambda MDSM49 differed from those of serotype g strain. The cell-surface protein antigen I/II from serotype c Streptococcus mutans is a potential immunogen for vaccination against dental caries and corresponds to the spaA from serotype d and g strains. A recombinant clone, pDM51, will be a useful tool for serological and molecular biological studies. The recombinant spaA provides useful material for assessment of its diagnostic and immunogenic potential.

Cloning of the gene encoding a glycylprolyl aminopeptidase from Porphyromonas gingivalis.

A genomic library of Porphyromonas gingivalis 381 was constructed in the cosmid vector pHC79. A clone, pSN1, was identified by the expression of glycylprolyl-naphthylamide hydrolysing activity. The DNA insert contained within the cosmid pSN1 was subcloned into the plasmid vector pBR328 to create the recombinant plasmid pSN11 containing a 2.9 kb EcoRV insert. An Escherichia coli transformant containing pSN11 produced a protein having a molecular weight of 75 kDa. Southern-blot hybridization revealed that the 2.9 kb EcoRV DNA hybridized with an identical sized Eco RV DNA fragment in the chromosomal DNA of P. gingivalis 381.

Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus.

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.

Cloning of a Bacteroides gingivalis outer membrane protein gene in Escherichia coli.

Gene banks of chromosomal DNA from Bacteroides gingivalis 381 were constructed using the bacteriophage replacement vector lambda L47.1. A clone encoding an outer membrane protein from B. gingivalis was identified by Western blot screening with antiserum raised against the outer membrane fraction of B. gingivalis 381 cells. The DNA insert contained within this phage was cloned into the plasmid vector pACYC184 to create the recombinant plasmid pMD123. An Escherichia coli transformant, MD123, containing pMD123 produced a protein having an apparent molecular weight of 40 kDa. The recombinant protein was purified, and amino acid analysis revealed the recombinant protein to have a relatively high content of hydrophobic amino acids (43.6%). Antiserum against the purified recombinant 40 kDa protein reacted with a polypeptide of similar size in the outer membrane fraction and vesicles of B. gingivalis.

Stimulation by interleukin-1 of interleukin-6 production by human periodontal ligament cells.

Interleukin-1(IL-1), a cytokine present in the gingiva and crevicular fluid of patients with periodontitis and in the periodontal ligament (PDL) of experimentally moved teeth, has multiple biological activities, including the ability to elicit bone resorption. Interleukin-6, also found in the gingiva of patients with periodontitis, may induce osteoclastic bone resorption through an effect on osteoclastogenesis. Here IL-6 production and its gene expression in response to recombinant IL-1 beta were examined in primary cultures of PDL cells. IL-1 beta stimulated IL-6 production by these cells in a dose- and time-dependent manner; this increase in IL-6 production was much higher than that in human gingival fibroblasts. In situ hybridization, using a synthetic oligonucleotide DNA probe of the IL-6 gene, revealed that most PDL cells expressed IL-6 mRNA in response to IL-1 beta treatment. The finding that IL-6 is produced by PDL cells and is regulated by IL-1 beta has revealed a potentially important mechanism for controlling alveolar bone resorption.

Some properties of prostaglandin E2 receptors in rabbit alveolar bone cell membranes.

[3H]prostaglandin E2 (PGE2) binding receptors exist in rabbit alveolar bone cell membranes. The presence of high (Kd = 3.9 X 10(-9) M) and low (Kd = 8.8 X 10(-8) M) affinity binding sites of [3H]PGE2 was demonstrated. The saturation values of [3H]PGE2 for high and low affinity binding sites were 0.13 pmol/mg protein and 1.22 pmol/mg protein, respectively. The digestion of the membranes with pronase, phospholipase C, D and neuraminidase led to a decrease of [3H]PGE2 binding but phospholipase A2 did not.