Tamaki Cho

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)

Mutation in IRO1 tightly linked with URA3 gene reduces virulence of Candida albicans.

Gene deletion in the pathogenic fungus Candida albicans has relied heavily on a URA3 cassette and a recipient Aura3 strain CAI4. The IRO1 gene adjacent to URA3 was inadvertently deleted during construction of CAI4. We report here that a mutation in IRO1 reduces virulence of C. albicans.

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.

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

JNK is critical for the development of Candida albicans-induced vascular lesions in a mouse model of Kawasaki Disease

BACKGROUND Kawasaki disease (KD) is the most common systemic vasculitis of unknown etiology in children, and can cause the life-threatening complication of coronary artery aneurysm. Although a novel treatment strategy for patients with KD-caused vascular lesions is eagerly awaited, their molecular pathogenesis remains largely unknown. c-Jun N-terminal kinase (JNK) is a signaling molecule known to have roles in inflammation and tissue remodeling. The aim of this study was to elucidate significant involvement of JNK in the development of vascular lesions in a mouse model of KD. METHODS AND RESULTS We injected Candida albicans cell wall extract (CAWE) into 4-week-old C57BL/6 mice. Macroscopically, we found that CAWE caused the development of bulging lesions at coronary artery, carotid artery, celiac artery, iliac artery and abdominal aorta. Histological examination of coronary artery and abdominal aorta in CAWE-treated mice showed marked inflammatory cell infiltration, destruction of elastic lamellae, loss of medial smooth muscle cells and intimal thickening, which are similar to histological features of vascular lesions of patients with KD. To find the role of JNK in lesion formation, we evaluated the effects of JNK inhibitor, SP600125, on abdominal aortic lesions induced by CAWE. Interestingly, treatment with SP600125 significantly decreased the incidence of lesions and also protected against vascular inflammation and tissue destruction histologically, compared with the placebo treatment. CONCLUSIONS Our findings suggest that JNK is crucial for the development of CAWE-induced vascular lesions in mice, and potentially represents a novel therapeutic target for KD.