Hidenori Kaminishi

Degradation of human dentine collagen by an enzyme produced by the yeast Candida albicans

An extracellular collagenolytic enzyme separated from a culture medium of this pathogenic yeast was found to attack undenatured predentine collagen as seen in scanning electron micrographs. After treatment with the enzyme at pH 4.0, but not by that acidity alone, dentine tubules were less easily distinguished and the collagen fibres were less well-organized.

A poly(γ-glutamic acid)–amphiphile complex as a novel nanovehicle for drug delivery system

Recently, many studies have focused on biomedical and pharmaceutical applications of self-assembled nanoparticles. In addition, several biodegradable nanoparticles have been reported to possess poor dispersion stability and poor size-controllability. However, these nanoparticles require complicated fabrication procedures using synthesis techniques. We developed an efficient method for producing nanoparticles derived from a biological origin of molecule poly(γ-glutamic acid) (γ-PGA), a cationic lipid, and doxorubicin (Dox). The complex had a size of 510 nm and was able to encapsulate over 90% of the added Dox. An in vivo assay of antitumor activity demonstrated that the complex had significant antitumor activity in sarcoma 180–bearing mice, and was effectively accumulated in solid tumors based on the EPR effect. The data suggested that this complex is a promising formulation of γ-PGA for targeted delivery to solid tumors. γ-PGA–12GP2 complexes may possess several unique advantages, including simplicity of nanoparticle preparation, high drug-carrying capacity, appropriate size to allow deeper penetration based on EPR effect into solid tumors, and lack of necessity to modify the chemical structure of the drugs. These data indicate that the γ-PGA–12GP2 complexes are potentially useful in cancer chemotherapy.

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)

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.

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.

Inflammatory Immune Responses by Water-insoluble α-glucans

Water-insoluble α-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 β-glucans in fungal cell wall components and water-soluble α-glucans from Streptococcus mutans are known to modulate innate immunity. In the present study, we investigated whether water-insoluble α-glucans also induced inflammatory innate immune responses. Our results showed that water-insoluble α-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 α-glucan, lipopolysaccharide, or peptidoglycan. Furthermore, human monocytes stimulated by water-insoluble α-glucans produced TNF-α and IL-8, while human polymorphonuclear cells were activated by water-insoluble α-glucans, resulting in chemotaxis and hydrogen peroxide production. The results demonstrated that water-soluble α-glucans modulate macrophage- and granulocyte-induced inflammatory immune responses, and suggest that inflammation induced by those α-glucans is associated with the development of periodontal diseases.

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.

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.

Isolation and sequencing of the Candida albicans MSI3, a putative novel member of the HSP70 family

We have reported previously that the expression of CGR1 increased at an early stage of the yeast–mycelial transition (morphogenesis) in Candida albicans. We now show that Cgr1p interacts in a yeast two‐hybrid system with the C. albicans Msi3p (CaMsi3p), a putative novel member of the heat shock protein 70 (HSP70) family. The DNA sequence of CaMSI3 encodes a predicted protein of 702 amino acids with a molecular mass of 78.6 kDa. The amino acid sequence of CaMsi3p is 63% identical to Msi3p/Sse1p of the HSP70 family of Saccharomyces cerevisiae. Further, CaMSI3 complemented the temperature‐sensitive phenotype of the msi3− mutant of S. cerevisiae. Other heat shock proteins of C. albicans are required for morphogenesis and are highly antigenic. These observations suggest that CaMSI3 may well provide functions for this organism unrelated to a heat shock function. The DDBJ Accession No. for the sequence reported in this paper is AB061274. Copyright

Candida albicans Msi3p, a homolog of the Saccharomyces cerevisiae Sse1p of the Hsp70 family, is involved in cell growth and fluconazole tolerance

We investigated the cellular function of Msi3p, belonging to the heat shock protein 70 family, in Candida albicans. The mutant strain tetMSI3 was generated, in which MSI3 was controlled by a tetracycline-repressive promoter, because there is evidence to suggest that MSI3 is an essential gene. We controlled the MSI3 expression level by doxycycline (DOX) and compared its phenotype with that of a control strain with the tetracycline-repressive promoter and a wild-type copy MSI3. The results indicated that MSI3 was essential for cell growth. In addition, all the tetMSI3-infected mice survived after DOX administration. Drug susceptibility tests indicated that repression of MSI3 expression resulted in hypersensitivity to fluconazole and conferred fungicidal activity to fluconazole. The expression levels of MSI3 and calcineurin-dependent genes were upregulated in response to fluconazole in the control strain. In tetMSI3, the upregulation of MSI3 was lost, and the expression level of the calcineurin-dependent genes was no longer elevated in response to fluconazole and was not affected by DOX, indicating that the upregulation of MSI3 expression was required for the induction of the calcineurin-dependent gene expression. These data suggest that Msi3p confers fluconazole tolerance by partially influencing the calcineurin signaling pathway and also other tolerance mechanisms.