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.

In vitro efficacy of continuous mild heat stress on the antifungal susceptibility of Candida albicans biofilm formation.

Elevation in the temperature induces heat stress to both host cells and the invading pathogen. This study aimed to determine whether continuous mild heat stress (increased temperature without causing significant damage to host cells) can increase susceptibility of biofilm formation of the opportunistic fungal pathogen Candida albicans to low concentrations of three typical antifungal agents. In this way the side effects associated with higher concentrations of the antifungal agents on host cells would be reduced. Fluconazole and micafungin at concentrations ranging from 0.0625 to 2 µg/mL and amphotericin B at concentrations ranging from 0.0625 to 1 µg/mL inhibited less than 20% of cells in biofilm formation. Biofilm formation at 39 or 41°C compared to 37°C resulted in increased susceptibility to the three agents, but especially micafungin. These data suggest that mild heat stress (39°C) would be valuable for increasing the effectiveness of low concentrations of antifungal agents against C. albicans biofilm formation. Thus, the concept of continuous mild heat stress at the site of insertion of medical devices or catheters combined with antifungal agents could be beneficial.

DNA/protamine complex paste for an injectable dental material.

A DNA/protamine complex powder was prepared by reaction between DNA and protamine sulfate solution with stirring in order to develop a new injectable biomaterials for dental therapy. The powder of DNA/protamine complex became paste by kneading the complex powder and distilled water. Complex formation was confirmed by FT-IR measurement. The complex paste had a porous structure and its viscosity was approximately 280.1 Pas. The paste could easily pass through a needle of 0.25 mm internal diameter. It seemed that DNA/protamine complex paste has suitable viscosity for clinical use as an injectable biomaterial. Although, the complex paste delayed the growth speed of Staphylococcus aureus, Pseudomonas aeruginosa, Porphyromonas gingivalis and Prevotella intermedia for limited periods, it cannot kill and inhibit growing bacteria. The complex paste disk showed a mild tissue response and gradually degraded after the implantation into the soft tissue of rats. These results suggested that this DNA/protamine complex paste could be a useful material for a biodegradable biomaterial. In particular, this paste will be applicable as an injectable biomaterial using syringe for the repair of defects of living tissue, GBR treatment and/or GTR treatment in dentistry.

Importance of Diversity in the Oral Microbiota including Candida Species Revealed by High-Throughput Technologies.

Taking advantage of high-throughput technologies, deep sequencing of the human microbiome has revealed commensal bacteria independent of the ability to culture them. The composition of the commensal microbiome is dependent on bacterial diversity and the state of the host regulated by the immune system. Candida species are well known as components of the commensal oral microbiota. Candida species frequently colonize and develop biofilms on medical devices like dentures and catheters. Therefore, Candida biofilm on dentures leads to a decrease in the bacterial diversity and then to a change in the composition of the oral microbiota. A disturbance in the balance between commensal bacteria and the host immune system results in a switch from a healthy state to a diseased state even in the limited oral niche.

Polycationic protamine for water-insoluble complex formation with DNA.

The DNA/protamine complex was prepared by a reaction between DNA and protamine sulfate solutions with stirring, and its cell viability, antibacterial effect and histopathological responses were examined. A water-insoluble white powder, DNA/protamine complex, with a porous structure was obtained. The molar binding ratio of the complex prepared from a solution containing equal amounts of DNA and protamine sulfate by weight was 0.038 and the efficiency of complex formation was 61%. In a cell culture test using MC-3T3-E1 mouse osteoblast cells, the complex showed less cytotoxicity than protamine sulfate alone and cell viabilities were more than 98%. A porous disk could be prepared easily and showed an antibacterial effect against Staphyrococcus aureus, Porphyromonas gingivalis and Prevotella intermedia in an antibacterial sensitivity test and a mild tissue response in vivo test. These results suggested that the DNA/protamine complex could be a useful biodegradable biomaterial with antibacterial effects.

A poly(gamma-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(gamma-glutamic acid) (gamma-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 gamma-PGA for targeted delivery to solid tumors. gamma-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 gamma-PGA-12GP2 complexes are potentially useful in cancer chemotherapy.