NO production in RAW264 cells stimulated with Porphyromonas gingivalis extracellular vesicles.

OBJECTIVE: This experiment was carried out in order to prove the inducible nitric oxide synthase (iNOS) expression and the nitric oxide (NO) production in mouse macrophage cells (RAW264) which were stimulated by vesicles released from Porphyromonas gingivalis, and discussed about the role of vesicles in advance periodontal diseases. MATERIALS AND METHODS: Production of NO(2) (-) in RAW264 cells was investigated after 0, 1, 3, 6 and 12h of stimulation with P. gingivalis vesicles. NO was analyzed by HPLC-based flow reactor system with Griess reagent. The cells stained by the enzyme-labeled antibody method, after being stimulated with vesicles for 12h. The iNOS proteins, which were expressed in RAW264 cells after 12h of stimulation with vesicles, were detected by western blot. RESULTS: When stimulated with vesicles from W83 and from ATCC33277, the RAW264 cells produced NO, but cell proteins that came in contact with the vesicles were degraded by protease activities in vesicles. When stimulated with vesicles from gingipain-deficient mutant strain KDP136, the RAW264 cells produced NO, but the quality was about 60%, compared with the vesicles from ATCC33277. CONCLUSION: The results suggest that vesicles are not only just a part of bacterial component, but also are a toxic complex of lipopolysaccharide and protease, and one of the putative virulence factor for periodontal diseases that continue inflammation and cause chronic conditions.

Inflammatory immune responses by water-insoluble alpha-glucans.

Water-insoluble alpha-glucans are synthesized from sucrose by glucosyltransferase-I of mutans streptococci and play an important role in the development of dental plaque. Several types of beta-glucans in fungal cell wall components and water-soluble alpha-glucans from Streptococcus mutans are known to modulate innate immunity. In the present study, we investigated whether water-insoluble alpha-glucans also induced inflammatory innate immune responses. Our results showed that water-insoluble alpha-glucans synthesized by Streptococcus sobrinus activated mouse peritoneal exudate macrophages to produce pro-inflammatory cytokines. The immunological responses were not due to contamination by sucrose, water-soluble alpha-glucan, lipopolysaccharide, or peptidoglycan. Furthermore, human monocytes stimulated by water-insoluble alpha-glucans produced TNF-alpha and IL-8, while human polymorphonuclear cells were activated by water-insoluble alpha-glucans, resulting in chemotaxis and hydrogen peroxide production. The results demonstrated that water-soluble alpha-glucans modulate macrophage- and granulocyte-induced inflammatory immune responses, and suggest that inflammation induced by those alpha-glucans is associated with the development of periodontal diseases.

Antifungal activity of DNA-lipid complexes and DNA-lipid films against Candida species.

In this study amphiphilic lipids, DNA-lipid complexes, and DNA-lipid films were prepared, and their antifungal activity against Candida species was examined. The amphiphilic lipids were synthesized from a reaction of glycine or L-alanine with n-alkyl alcohol in the presence of p-toluene sulfonic acid. DNA-lipid complexes, which were prepared by the simple mixing of DNA and amphiphilic lipids, were insoluble in water. Self-standing, water-insoluble DNA-lipid films were prepared by casting the DNA-lipid complexes from a chloroform/ethanol solution. The antifungal activities of the lipids and DNA-lipid complexes against the Candida species were evaluated by minimum inhibitory concentrations (MICs); those of DNA-lipid films were evaluated by the disk diffusion method. The seven kinds of lipids, DNA-lipid complexes, and DNA-lipid films showed antifungal activity, and no differences were seen in the antifungal activities between glycine and L-alanine derivatives. The lipids, DNA-lipid complexes, and DNA-lipid films, which have shorter alkyl chain length in lipids, showed antifungal activity against all Candida species. However, the effect of antifungal activity against Candida species decreased with increased alkyl chain length in lipids. In this study, it was found that lipids, DNA-lipid complexes, and films with a decyl or dodecyl group exhibit more favorable antifungal activity.

Antibacterial characteristics of newly developed amphiphilic lipids and DNA-lipid complexes against bacteria.

The purpose of this study was to investigate the antibacterial activity of newly developed amphiphilic lipids and DNA/lipid complexes against two types of oral bacteria and two types of hospital infection bacteria. Nine amphiphilic lipids were quantitatively prepared from the reaction of n-alkyl alcohol, alpha-amino acids, and p-toluenesulfonic acid. Nine DNA-lipid complexes were prepared by the simple mixing of DNA and amphiphilic lipids. The DNA-lipid complexes were insoluble in water. The antibacterial activity of lipids and DNA-lipid complexes against Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa were evaluated by the disk-diffusion method. Seven artificial lipids showed antibacterial behavior; in particular, the lipids prepared from n-decyl alcohol and glycine and from n-decyl alcohol and L-alanine showed antibacterial activity against the four bacterial strains used in this study. On the other hand, the lipids of glutamic acid derivatives did not show any antibacterial activity against the four bacteria strains except for the lipid with an n-octyl group. Five DNA-lipid complexes also had an antibacterial effect. The complex prepared from DNA and glycine decyl ester p-toluenesulfonic acid salt exhibited antibacterial activity against the four types of bacteria strains. In this study it was found that lipids and DNA-lipid complexes with a mono-decyl group or a mono-dodecyl group have more favorable antibacterial activity.

Isolation and expression of a gene (CGR1) regulated during the yeast-hyphal transition in Candida albicans.

We used RNA fingerprinting of arbitrarily primed PCR to isolate genes upregulated during the yeast-hyphal transition in Candida albicans. The sequence and expression of one of these genes (CGR1, Candida growth regulation) are presented. Our results suggest that CGR1 expression is associated with a growth cessation of yeast cells, a prerequisite for germination in this organism.

Inhibitors of protein phosphorylation including the retinoblastoma protein induce germination of Candida albicans.

It has been previously shown that the induction of germination in Candida albicans occurs following its cessation of growth as a yeast. Similarly, mammalian cells undergo a differentiation process that is preceded by a growth cessation associated with a hypophosphorylation of proteins of the retinoblastoma gene family. It is postulated that a similar type of mechanism may be operative in C. albicans and protein phosphorylation inhibitors: forskolin (stimulates cyclic adenosine monophosphate production), okadaic acid (phosphatase inhibitor) and D-erythro-sphingosine (retinoblastoma protein phosphorylation inhibitor) have been used to further strengthen this hypothesis. Okadaic acid (1-1000 nM) and D-erythro-sphingosine (100 microM) significantly inhibited the growth of yeast cells of C. albicans. D-Erythro-sphingosine at 1000 microM was candidicidal. Forskolin did not significantly affect growth. Exponentially grown C. albicans pretreated with forskolin (10 microM), okadaic acid (1000 nM) or D-erythro-sphingosine (100 microM) readily germinated. In comparison, when these inhibitors were incorporated in the same medium, germination of exponentially grown cells did not occur. These results suggest that protein dephosphorylation may be necessary at an early stage of the yeast-hyphae transition in C. albicans.

Candida albicans: adherence, signaling and virulence.

The focus of this symposium was to present new information on the morphogenesis of Candida albicans, particularly how it relates to signal transduction pathways and other genes involved in the regulation of morphogenesis. In addition, we discuss the role of adherence and colonization of the oral cavity by the organism and discuss the role of mannan as an adhesin that recognizes the human red blood cell. C. albicans utilizes at least two signal pathways to regulate its conversion from a yeast form to filamentous growth (hyphae). One of these two pathways is similar to the Saccharomyces cerevisiae pseudohyphal/mating pathway, which utilizes the regulatory protein, Cphlp. The other pathway is not totally defined but requires a second regulatory protein, referred to as Efg1p. Other signal pathways may exist, which include a two-component histidine kinase and response regulator proteins. The latter pathway(s) may include proteins such as Chk1p, Ssk1p, Shi1p and Cos1p/Nik1p. Mutations in strains, which specifically target these proteins, result in morphogenesis defects and avirulence or attenuation of strains. A growth regulatory gene has also been recently defined whose expression is associated with growth cessation and which appears to be a necessary prerequisite in conversion of the organism to a filamentous growth form. Starvation of yeast cells induces exponentially grown cells (and usually non-germinative) to germinate. This phenomenon is also observed in cells that are transiently treated with metabolic inhibitors. During each of these treatments (starvation, metabolic inhibition), expression of a growth regulatory gene (CGRI) increases. Adherence of C. albicans to host cells and tissues is complex; several proteins, which appear to have host recognition functions, have been defined. In the oral cavity, C. albicans selectively adheres to salivary proteins, which are absorbed to many oral surfaces. This mechanism enables the cells to colonize surfaces of the oral cavity. An understanding of these interactions may lead to strategies to prevent oral disease. Mannan from C. albicans may provide a host recognition function for C. albicans. Recent experiments indicate that mannan binds to human red blood cells and causes hemolysis. Binding of mannan to the band 3 protein of human red blood cells has been established. This activity may be associated with the ability of the organism to utilize hemoglobin (and iron).

Degradation of humoral host defense by Candida albicans proteinase.

The effect of an extracellular proteinase from the pathogenic yeast Candida albicans on the bactericidal and opsonizing activities of human serum was studied. The ability of human polymorphonuclear leukocytes to kill Staphylococcus aureus was greatly reduced when the bacteria were opsonized with human serum treated with the proteinase. The reduction in the opsonizing activity of human serum was attributed to degradation of the Fc portion of immunoglobulin G by the action of C. albicans proteinase as determined by immunoprecipitation reaction. However, the Fab portion of immunoglobulin G was resistant to proteolysis by the proteinase. A clear reduction in the bactericidal activity of human serum against Escherichia coli was observed when the serum was treated with C. albicans proteinase. The reduction of serum bactericidal activity was attributed to the degradation of complement C3 by proteolysis by the proteinase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while C5 resisted the action of the proteinase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteinase also degrades endogenous proteinase inhibitors, such as alpha 2 macroglobulin and alpha 1 proteinase inhibitor, which are involved in regulating inflammation. These results suggest that destruction of a host's defense-oriented or regulatory proteins facilitates debilitation of the infected host.

The relationship between the glucose uptake system and growth cessation in Candida albicans.

It is thought that dimorphic Candida albicans undergoes changes in its intracellular metabolic state prior to yeast-mycelial transformation. Cells grown in budding form to mid-exponential phase could not be induced to form germ tubes when grown in glucose medium. However, cells in which growth was initially inhibited by either starvation or inhibitors (0.1% hydroxyurea, 4% sodium malonate or 4% 2-deoxy-D-glucose) could be induced to form germ tubes in the same medium. The effects of these initial treatments on the intracellular state in mid-exponential phase cells were analysed by measuring the kinetics of D-glucose uptake. D-glucose uptake in mid-exponential phase and stationary phase cells was measured. The untreated mid-exponential phase cells exhibited only a high Km (6.9 mM). However, mid-exponential phase cells, in which growth was initially inhibited, exhibited both a high Km (3.2-6.2 mM) and a low Km (0.40-0.78 mM) simultaneously. In addition, the stationary phase cells exhibited both a high Km (5.6 mM) and a low Km (0.56 mM). These results suggest that there are two kinetically distinct systems of glucose transport in C. albicans and that changes in the glucose uptake system in C. albicans may be related to intracellular changes prior to transition from the budding to the mycelial form.

Spiral hyphae of Candida albicans formed in anaerobic culture.

Candida albicans from a patient with dental caries grew on minimal medium consisting of agar supplemented with magnesium chloride and sodium phosphate. Hyphal growth was observed when the yeast was cultured between 26 degrees C and 28 degrees C under aerobic conditions, and typical chlamydospores were formed. However, when the yeast was cultured at the same temperature under anaerobic conditions, curly hyphae developed on the surface of the medium, but no chlamydospores were formed. This phenomenon was also observed if the culture was started under aerobic conditions but was continued under anaerobic conditions.

Activation of blood clotting factors by microbial proteinases.

There are very few reports on the involvement of bacterial proteinases on the blood clotting system using both human plasma and purified clotting factors. We studied whether microbial proteinases from the opportunistic pathogens Candida albicans, Pseudomonas aeruginosa and Serratia marcescens activate the blood clotting cascade by using normal human plasma, human plasmas deficient in clotting factor XII or X, and also by using purified clotting factors XII, X and prothrombin. All proteinases tested activated either clotting factor XII or prothrombin in vitro, thus resulting in generation of thrombin. Clotting factor X was converted to the active form (Xa) by both Candida and Pseudomonas proteinases, but not by Serratia proteinase. These results suggest that peripheral and systemic blood circulation may be impaired by activation of the blood clotting cascade by microbial infections, especially in septic patients, which would enhance disseminated intravascular coagulation and multi-organ failure.

Bradykinin-B2 receptors in humans and rats: cDNA structures, gene structures, possible alternative splicing, and homology searching for subtypes.

1. To identify and isolate cDNAs encoding rat and human bradykinin-B2 receptor subtypes we isolated a human bradykinin receptor cDNA homologous to a rat B2 receptor cDNA. 2. The cDNA was expressed in the bradykinin receptor negative cell line, CHO; membranes prepared from these cells bound bradykinin and had specificity similar to that of the known rat B2 receptor. In addition, the expressed receptor has a low affinity for des-Arg9-bradykinin. Thus, the cDNA encodes a human B2-bradykinin receptor. 3. Comparison of the human and rat cDNAs suggested that the human and rat genes are composed of three exons. Cloning, sequencing and characterization of parts of the human and rat B2-bradykinin receptor genes demonstrated the postulated three-exon structure. This structure includes two 5' exons upstream of the most favorable translation initiation methionine in exon-3. 4. The two 5' exons each contain methionines, which if independently spliced to the third exon, would yield an open reading frame that includes all of exon-3. This arrangement could thus vary the amino-terminal region of the protein. Do these potential arrangements occur in human RNAs, and will they lead to proteins with differing amino-termini? 5. Reverse transcriptase-polymerase chain reactions (RT-PCR) using human mRNA, nested primers from exon-1 and exon-3, and detection of the products by hybridization using an independent exon-1 oligonucleotide showed that the arrangement of exon-1 with exon-2 and exon-3 could not be detected in eight human RNAs. Furthermore, exon-1 spliced with exon-3 was a common arrangement. 6. Low stringency examination of human and rat Southern blots revealed only bands attributable to the known human or rat B2-bradykinin receptor. 7. Reduced stringency hybridization searches of seven different genomic and cDNA libraries--including two different human genomic libraries, a rat genomic library, two different rat uterus cDNA libraries, a rat brain library and a human lung library--yielded only rat or human B2-bradykinin receptors. The results of our low stringency hybridization experiments suggest that other bradykinin receptors are less than 60% identical, on the nucleotide level, to the known B2 receptor. 8. Degenerate polymerase chain reactions using rat genomic DNA as a template and degenerate primers, designed based on the homology of a B2-bradykinin receptor with angiotensin-II type-1 receptor, identified B2-bradykinin receptors, angiotensin-II-type-1 receptors and three novel orphan receptors.

Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs.

Porphyromonas gingivalis protease, which had been isolated from a culture supernatant, caused vascular permeability enhancement in a dose-dependent manner when injected into guinea pig skin. The permeability-enhancing reaction caused by the protease was not affected by treatment with antihistamine, but was greatly augmented by simultaneous injection of a kinin potentiator, carboxypeptidase N inhibitor. However, the reaction was inhibited by soybean trypsin inhibitor or alpha 2-antiplasmin, although both of these inhibitors could not inhibit P. gingivalis protease at all by themselves. A bradykinin-degrading enzyme, carboxypeptidase B, weakened this vascular reaction. Results described indicate that the permeability-enhancing reaction induced by the protease is caused by activation, of the kallikrein-kinin cascade in the tissue.

The relationship between cyclic adenosine 3',5'-monophosphate and morphology in exponential phase Candida albicans.

The relationship between changes in cyclic AMP content and germ tube formation in exponential phase Candida albicans was investigated using two simple media containing glucose plus ammonium chloride, or N-acetyl-D-glucosamine (GlcNAc). The glucose medium did not promote germ tube formation unless the cells were starved before inoculation, whereas the GlcNAc medium promoted germ tube formation in both non-starved and starved cells. The cyclic AMP content of exponential phase cells, non-starved cells and starved cells was 0.21, 0.34 and 0.64 pmol mg-1 dry wt, respectively. In glucose medium, cyclic AMP content in both non-starved cells and starved cells increased for a period of 60 min after inoculation, but then decreased for a further 120 min. The cyclic AMP content of non-starved cells and starved cells was 0.16 and 0.29 pmol mg-1 dry wt, respectively, after 180 min. The maximum percentage of non-starved cells with germ tubes was around 20%. Starved cells with germ tubes were observed after 40 min and reached a maximum (around 90%) after 140 min. The number of germ tubes remained constant for the next 40 min. In GlcNAc medium, the cyclic AMP content of both non-starved cells and starved cells showed a tendency to increase for 180 min. The content of non-starved cells and starved cells was 2.02 and 1.75 pmol mg-1 dry wt, respectively, after 180 min. Germ tube formation in non-starved cells started after 70 min, reached around 80% after 150 min, and remained stable for the next 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the plasma kallikrein-kinin system by Candida albicans proteinase.

An extracellular carboxyl proteinase produced by the yeast Candida albicans enhanced vascular permeability when injected into the dorsal skin of guinea pigs. The character and mechanism of the permeability-enhancing reaction were studied in vivo and in vitro. Permeability was not enhanced when the C. albicans proteinase was heat treated (100 degrees C, 5 min) or when it was treated with pepstatin, a specific carboxyl proteinase inhibitor. The permeability reaction induced by the C. albicans proteinase was not affected by pretreatment with antihistamine but was greatly augmented by simultaneous injection of a kinin potentiator, carboxypeptidase N inhibitor. However, the simultaneous injection of a kinin-degrading enzyme, carboxypeptidase B, interfered with the reaction. Furthermore, in vitro conversion of plasma prekallikrein to kallikrein by the C. albicans proteinase was observed, and the reaction was inhibited by corn trypsin inhibitor, an inhibitor of activated Hageman factor, and soybean trypsin inhibitor, a well-known inhibitor of plasma kallikrein. These results indicate that C. albicans proteinase enhances vascular permeability through activation of the plasma kallikrein-kinin system, which generates bradykinin.

Starvation and germ tube formation in the exponential phase Candida albicans.

Two chemically defined media were developed for the induction of germ tubes in exponential phase cells of Candida albicans. One medium was N-acetyl-D-glucosamine medium which is composed of L-thiazolidine-4-carboxylic acid, L-proline, NaHCO3, sodium acetate, NaH2PO4 and N-acetyl-D-glucosamine. The other one was glucose medium in which N-acetyl-D-glucosamine is exchanged for glucose plus NH4Cl in N-acetyl-D-glucosamine medium. In these media, a high percentage of germ tube forming cells was obtained without a temperature shift. However, starvation of the cells in water at 37 degrees C was a necessary pretreatment to consistently obtain a high percentage of germ tube forming cells. The effect of starvation was remarkable in glucose medium, the percentages of germ tube forming cells among the normal cells and starved cells were 20 and 80, respectively. As for intracellular changes during starvation, a decrease in adenosine triphosphate concentration and an increase in adenosine 3',5'-cyclic monophosphate concentration were observed.

Degradation of bovine achilles tendon collagen by Candida albicans proteinase.

Candida albicans, when cultivated in a medium containing insoluble bovine achilles tendon as a nitrogen source, was able to produce a collagen degrading proteinase. The degradation of achilles tendon collagen by the proteinase was verified by morphological change and the release of hydroxyproline. The proteinase activity was inhibited by pepstatin.