Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.     

Isolation of porphyran-degrading marine microorganisms from the surface of red alga, Porphyra yezoensis

Marine microorganisms degrading porphyran (POR) were found on the surface of thalli of Porphyra yezoensis. Fifteen crude microorganism groups softened and liquefied the surface of agar-rich plate medium. Among these, 11 microorganism groups degraded porphyran that consisted of sulfated polysaccharide in Porphyra yezoensis. Following isolation, 7 POR-degradable microorganisms were isolated from the 11 POR-degradable microorganism groups.     

Regulation of the body fat percentage in developmental-stage rats by methylxanthine derivatives in a high-fat diet

We investigated the regulatory effects of structural differences among methylxanthine derivatives on the elevation of body fat percentage in developmental-stage rats. Caffeine, theophylline and theobromine were used as the methylxanthines. High-fat diets (20% lard) containing each methylxanthine (0.025%) were administered to male Sprague-Dawley rats for 12 weeks, with the result that the body fat percentage was generally reduced in each methylxanthine-fed group. The abdominal adipose tissue weight in the caffeine group was also significantly lower than that in the control group, the serum cholesterol and triglyceride levels in the caffeine group also being significantly lower than the levels in the control group. The study results suggest that caffeine could contribute most to preventing arteriosclerotic diseases.     

Solution structure and binding specificity of FBP11/HYPA WW domain as Group-II/III

The Group-II/III WW domains bind Pro-rich sequences, the most frequent protein motif found in eucaryotic genomes. We have proposed that the Group-II and -III WW domains be merged into a larger group because the members of each group have relatively wide specificity and bind to the common ligands [Kato et al., J Biol Chem 2004;279:31833-31841]. We have also proposed that Group-II/III has a common surface patch, the XP2 groove, to bind the ligands. The first WW domain of FBP11/HYPA is one of the Group-II/III WW domains. The solution structure of the 26 residue-long converged region exhibits an antiparallel triple stranded beta-sheet with a small hydrophobic core. The WW domain of FBP11/HYPA has both XP and XP2 grooves on its surface. Ligand titration by 1H-15N HSQC NMR spectra revealed that the WW domain of FBP11/HYPA binds all the peptides with the PL, PP, and PR motifs. The profile patterns of chemical shift perturbation were quite similar among the spectra titrated with all three ligands. In addition, the titration significantly shifts the signals of the residues that compose the XP2 groove. All these findings suggest the functional importance of the XP2 groove and group definition of Group-II/III of the WW domains.     

Effects of peripheral cannabinoid receptor ligands on motility and polarization in neutrophil-like HL60 cells and human neutrophils

The possible role of the peripheral cannabinoid receptor (CB2) in neutrophil migration was investigated by using human promyelocytic HL60 cells differentiated into neutrophil-like cells and human neutrophils isolated from whole blood. Cell surface expression of CB2 on HL60 cells, on neutrophil-like HL60 cells, and on human neutrophils was confirmed by flow cytometry. Upon stimulation with either of the CB2 ligands JWH015 and 2-arachidonoylglycerol (2-AG), neutrophil-like HL60 cells rapidly extended and retracted one or more pseudopods containing F-actin in different directions instead of developing front/rear polarity typically exhibited by migrating leukocytes. Activity of the Rho-GTPase RhoA decreased in response to CB2 stimulation, whereas Rac1, Rac2, and Cdc42 activity increased. Moreover, treatment of cells with RhoA-dependent protein kinase (p160-ROCK) inhibitor Y27632 yielded cytoskeletal organization similar to that of CB2-stimulated cells. In human neutrophils, neither JWH015 nor 2-AG induced motility or morphologic alterations. However, pretreatment of neutrophils with these ligands disrupted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced front/rear polarization and migration and also substantially suppressed fMLP-induced RhoA activity. These results suggest that CB2 might play a role in regulating excessive inflammatory response by controlling RhoA activation, thereby suppressing neutrophil migration.     

IgE-dependent activation of sphingosine kinases 1 and 2 and secretion of sphingosine 1-phosphate requires Fyn kinase and contributes to mast cell responses

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells results in the production and secretion of sphingosine 1-phosphate (S1P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of sphingosine kinase (SphK1 and SphK2) are expressed and activated upon FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI coupling to SphK1 and -2 and for subsequent S1P production. Normal activation of SphK1 and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK1 and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK1 activation differed from that of SphK2. Whereas SphK1 was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S1P to FcepsilonRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.     

Complement-mediated phagocytosis--the role of Syk

Phagocytosis is a central event in the innate immune responses that are triggered by the association between ligands on the surface of pathogens and receptors on the membrane of phagocytes. Particularly, complement-mediated phagocytosis is accomplished by specific recognition of bound complement components by the corresponding complement receptors on the phagocytes. The protein-tyrosine kinase, Syk, plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. From recent studies using a macrophage-like differentiated cell line and serum-treated zymosan, it was found that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Serum-treated zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor3. During this process, Syk became tyrosine-phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into macrophages resulted in impaired engulfment of pathogen. Collectively, Syk is required for the engulfment of pathogen in complement-mediated phagocytosis.     

Ascochlorin inhibits growth factor-induced HIF-1α activation and tumor-angiogenesis through the suppression of EGFR/ERK/p70S6K signaling pathway in human cervical carcinoma cells

Ascochlorin, a non-toxic prenylphenol compound derived from the fungus Ascochyta viciae, has been shown recently to have anti-cancer effects on various human cancer cells. However, the precise molecular mechanism of this anti-cancer activity remains to be elucidated. Here, we investigated the effects of ascochlorin on hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human epidermoid cervical carcinoma CaSki cells. Ascochlorin inhibited epidermal growth factor (EGF)-induced HIF-1α and VEGF expression through multiple potential mechanisms. First, ascochlorin selectively inhibited HIF-1α expression in response to EGF stimulation, but not in response to hypoxia (1% O(2)) or treatment with a transition metal (CoCl(2)). Second, ascochlorin inhibited EGF-induced ERK-1/2 activation but not AKT activation, both of which play essential roles in EGF-induced HIF-1α protein synthesis. Targeted inhibition of epidermal growth factor receptor (EGFR) expression using an EGFR-specific small interfering RNA (siRNA) diminished HIF-1α expression, which suggested that ascochlorin inhibits HIF-1α expression through suppression of EGFR activation. Finally, we showed that ascochlorin functionally abrogates in vivo tumor angiogenesis induced by EGF in a Matrigel plug assay. Our data suggest that ascochlorin inhibits EGF-mediated induction of HIF-1α expression in CaSki cells, providing a potentially new avenue of development of anti-cancer drugs that target tumor angiogenesis.     

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells

The HSP30 gene of the budding yeast Saccharomyces cerevisiae encodes a seven-transmembrane heat shock protein expressed in response to various types of stress including heat shock. Although Hsp30p contains a potential N-glycosylation consensus sequence (Asn(2)-Asp(3)-Thr(4)), whether it is actually N-glycosylated has not been verified. Here we demonstrate that N-glycosylation is induced at Asn(2) of Hsp30p by severe heat shock, ethanol stress, and acetic acid stress. Mild heat shock and glucose depletion induced the expression but not N-glycosylation of Hsp30p, indicating the N-glycosylation to be dependent on temperature and environmental conditions. N-glycosylation did not affect on the intracellular localization of Hsp30p but its physiological role under severe heat shock conditions. Since limited information is available on stress-responsive or condition-induced N-glycosylation, our findings provide new insight into the regulation of cellular stress response in yeast.     

Copy number of adenoviral vector genome transduced into target cells can be measured using quantitative PCR: application to vector titration

Both transfection and adenovirus vectors are commonly used in studies measuring gene expression. However, the real DNA copy number that is actually transduced into target cells cannot be measured using quantitative PCR because attached DNA present on the cell surface is difficult to distinguish from successfully transduced DNA. Here, we used Cre/loxP system to show that most of the transfected DNA was in fact attached to the cell surface; in contrast, most of the viral vector DNA used to infect the target cells was present inside the cells after the cells were washed according to the conventional infection protocol. We applied this characteristic to adenoviral vector titration. Current methods of vector titration using the growth of 293 cells are influenced by the effect of the expressed gene product as well as the cell conditions and culture techniques. The titration method proposed here indicates the copy numbers introduced to the target cells using a control vector that is infected in parallel (relative vector titer: rVT). Moreover, the new titration method is simple and reliable and may replace the current titration methods of viral vectors.