The antibacterial activity of fluoroquinolones against gram-negative bacteria isolated from Gifu Prefecture (2005)

We investigated the susceptibility to 6 fluoroquinolones against 433 strains of Gram-negative bacteria isolated from 6 medical facilities in Gifu prefecture between January and September in 2005, determined by the agar dilution methods in according with the Japan Society of Chemotherapy. We also investigated the correlation between the degree of resistance to fluoroquinolones and the amino acid substitutions in the quinolone resistance-determining region (QRDR). The tested clinical isolates were as follows, Salmonella spp.; 17 strains, Escherichia coli; 112 strains Citrobacter freundii; 35 strains, Enterobacter cloacae; 31 strains, Klebsiella pneumoniae; 73 strains, Proteus spp.; 18 strains, Providencia spp.; 3 strains, Morganella morganii; 14 strains, Serratia marcescens; 27 strains and Pseudomonas aeruginosa; 103 strains. The number of the strains resistant to ciprofloxacin (CPFX) (MIC > or = 6.25 microg/mL) was twenty (E. coli; 14 strains, E. cloacae; I strain, Proteus spp.; 2 strains and P. aeruginosa; 3 strains). Among these strains, 12 strain (E. coli; 11 strains and E. cloacae; 1 strain) were highly resistant to CPFX (MIC > or =25 microg/mL). The E. coli strains highly resistant to CPFX had the multiple amino acid mutations in QRDR of ParC an GyrA. However in other strains, there was no strains possessing multiple mutations in both ParC and GyrA.     

Kojic acid -absence of tumor-initiating activity in rat liver, and of carcinogenic and photo-genotoxic potential in mouse skin

Kojic acid (KA) has been widely used as a quasi-drug ingredient. Possible promotion activity of KA was suggested on livers of mouse and rat by findings obtained in genotoxicity and carcinogenicity studies performed thus far. Therefore, in order to examine safety as a quasi-drug ingredient, we investigated the presence of initiation activity in rat liver and the photo-genotoxicity and carcinogenicity in mouse skin. In medium-term carcinogenesis test in rats, 2.0% KA was orally given to F344/DuCrj rats for 4 weeks of the initiation period, followed by the combination of partial hepatectomy and treatment with a hepatocarcinogenesis promoter, phenobarbital (PB). As a result, glutathione S-transferase placental form (GST-P) positive foci of 0.2 mm or more in diameter in the KA group, which is usually used in determination of pre-cancerous lesions, did not increase significantly in both numbers and areas compared with those of the non-initiated controls. In the concurrent analysis, however, numbers of GST-P-positive foci of two cells or more and 0.1 mm or more in diameter increased slightly, and possible weak initiation activity of KA was equivocal. However, considering the known fact that KA exerts promotion activity in the liver of F344 rats by long-term dietary administration, it was suggested that the observed slight increase of the numbers of GST-P-positive foci in rat liver was the effect of promotion activity of KA rather than the initiation activity. In DNA adducts formation assay in a rat liver, no clear adducts derived from KA were detected in male F344/DuCrj rats administered 0.5% or 2% KA orally, and KA was considered not to form DNA adducts in rat liver. In the in vitro photo-reverse mutation assay with bacteria, KA exerted weak photo-mutagenicity. Furthermore, in chromosome aberration study in Chinese hamster lung cells (CHL/IU cells) with UV irradiation, KA induced chromosome aberration at high-dose (1.4 mg/mL) treatment with UV irradiation, but was negative without UV irradiation. However, in the in vivo photo-micronucleus study in mouse, in which 1.0 or 3.0% KA containing cream was applied twice to the back of the animals with a 24-hr interval, KA did not induce micronuclei in mouse epidermal cells. Based on these results, it is considered that the risk of KA to exert photo-carcinogenicity is quite low in the skin. In skin carcinogenesis bioassay for initiation-promotion potential, 3.0% KA cream formulation was applied to the back of the mouse for 1 week (once a day, total 7 times) and for 19 weeks (5 times a week, total 95 times) during the initiation and the promotion stages, respectively. No skin nodules were observed in any animal skins formed due to KA treatment given in either stage. Therefore, KA is considered not to possess initiation nor promotion activity of skin carcinogenesis. Furthermore, from the above findings, it is suggested that KA is virtually safe as a quasi-drug ingredient.     

Creation of novel cell-penetrating peptides for intracellular drug delivery using systematic phage display technology originated from Tat transduction domain

Many biologically active proteins need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm. Cell penetrating peptides (CPPs) have been developed to efficiently deliver a wide variety of cargo in a fully biological active form into a range of cell types for the treatment of multiple preclinical disease models. To further develop this methodology, we established a systematic approach to identify novel CPPs using phage display technology. Firstly, we screened a phage peptide library for peptides that bound to the cell membrane. Secondly, to assess functionality as intracellular carriers, we recombined cDNAs of binding peptides with protein synthesis inhibitory factor (PSIF) to create fusion proteins. Randomly chosen clones were cultured and expression of peptide-PSIF fusion proteins induced, followed by screening of protein synthesis activity in cells. Using this systematic approach, novel and effective CPPs were rapidly identified. We suggest that these novel cell-penetrating peptides can utilized as drug delivery tools for protein therapy or an analytical tool to study mechanisms of protein transduction into the cytoplasm.     

Prostate cancer diagnosed through the biopsy of the bone metastatic lesion; a case report

An 80-year-old man visited our clinic with the chief complaint of asymptomatic macroscopic hematuria secondary to anticoagulant medicine. Although digital rectal examination was normal, a high serum prostate specific antigen (PSA) level (85.9 ng/ml) led us to perform sextant prostate biopsy, resulting in negative for cancer. Three months later, since the serum PSA increased to 169 ng/ml with high serum carcinoembryonic antigen (CEA) and carbohydrate antigen (CA) 19-9 levels. Twelve-core prostate biopsy was performed again, but the result was negative. Pelvic magnetic resonance imaging (MRI) showed a metastatic lesion on the right pubic bone, which was biopsied, and turned out to be poorly differentiated prostate cancer in histology. Maximum androgen blockade failed to control PSA. Finally he died of pneumonia 55 days after the bone biopsy. To our knowledge, there were only two case reports diagnosed as prostate cancer by biopsies of the metastatic lesions in Japanese literature, but none in the English literature. These findings suggest that high serum levels of CEA and CA19-9 in patients with prostate cancer are indications of hormone-refractory prostate cancer resulting in poor prognosis.     

Maternal exposure to anti-androgenic compounds, vinclozolin, flutamide and procymidone, has no effects on spermatogenesis and DNA methylation in male rats of subsequent generations

To verify whether anti-androgens cause transgenerational effects on spermatogenesis and DNA methylation in rats, gravid Crl:CD(SD) female rats (4 or 5/group, gestational day (GD) 0 = day sperm detected) were intraperitoneally treated with anti-androgenic compounds, such as vinclozolin (100 mg/kg/day), procymidone (100 mg/kg/day), or flutamide (10 mg/kg/day), from GD 8 to GD 15. Testes were collected from F1 male pups at postnatal day (PND) 6 for DNA methylation analysis of the region (210 bp including 7 CpG sites) within the lysophospholipase gene by bisulfite DNA sequencing method. F0 and F1 males underwent the sperm analysis (count, motility and morphology), followed by DNA methylation analysis of the sperm. Remaining F1 males were cohabited with untreated-females to obtain F2 male pups for subsequent DNA methylation analysis of the testes at PND 6. These analyses showed no effects on spermatogenesis and fertility in F1 males of any treatment group. DNA methylation status in testes (F1 and F2 pups at PND 6) or sperms (F1 males at 13 weeks old) of the treatment groups were comparable to the control at all observation points, although DNA methylation rates in testes were slightly lower than those in sperm. In F0 males, no abnormalities in the spermatogenesis, fertility and DNA methylation status of sperm were observed. No transgenerational abnormalities of spermatogenesis and DNA methylation status caused by anti-androgenic compounds were observed.     

Oral flora in independent over 80-year-olds with more than 20 teeth

The purpose of this study was to investigate oral flora in independent persons aged over 80 years with more than 20 remaining teeth. The subjects were 22 participants of the 8020 campaign (6 males and 16 females) with a mean age of 81.3+/-1.6 years and an average of 24.7 teeth (Independent 8020 group). This group was compared with a group of 38 elderly people residing in nursing homes (10 males and 28 females) who had a mean age of 81.3+/-8.5 years and an average of 4.2 teeth (Nursing group with fewer teeth). Saliva samples were collected from the vestibular areas of the maxilla and mandible using cotton swabs. Cell numbers of microorganisms were expressed as colony forming units/ml (CFUs/ml) and compared between the two groups. The average number of Staphylococcus species was 65.2+/-74.4 CFUs/ml in the Independent 8020 group and 400.3+/-352.1 CFUs/ml in the group with fewer teeth (p<0.01); that of Candida albicans was 18.0+/-37.7 CFUs/ml in the Independent 8020 group and 152.9+/-211.9 CFUs/ml in the Nursing group with fewer teeth (p<0.05). Both species showed statistically significant differences between the two groups. This suggests that the Independent 8020 achiever group had better oral hygiene and that the presence of many teeth may be associated with an increased awareness of dental health.     

Repeated intravenous injection of recombinant human hepatocyte growth factor ameliorates liver cirrhosis but causes albuminuria in rats

Hepatocyte growth factor (HGF) is a promising agent for the treatment of liver cirrhosis because of its mitogenic and anti-fibrotic effects. We investigated the effect of recombinant human HGF (rh-HGF) on cirrhosis development; its pharmacokinetics and nephrotoxicity in rats with liver cirrhosis induced by 4-week treatment with dimethylnitrosamine (DMN). rh-HGF (0.3 mg/kg) was intravenously administered to rats once a day for 4 weeks in parallel with DMN treatment or twice a day for the last 2 weeks of DMN treatment. Repeated doses of rh-HGF increased the liver weight and serum albumin, and reduced serum ALT. The development of hepatic fibrosis was inhibited more efficiently by extended low-dose treatment with rh-HGF. In cirrhotic rats, serum levels of rh-HGF increased and clearance was decreased, leading to an increase in the area under the plasma-concentration time curve and a decrease in the steady-state volume of distribution. Repeated doses of rh-HGF led to increased urinary albumin excretion, but no rh-HGF-treated animals developed increased serum creatinine levels. Urinary albumin excretion returned to baseline after the cessation of rh-HGF. These results suggest that extended treatment with rh-HGF is required for the attenuation of cirrhosis, and repeated doses of rh-HGF cause adverse effects in extra-hepatic organs. These issues must be resolved before the widespread application of rh-HGF in the treatment of liver cirrhosis.     

Side chain orientation of the amino acid substituted by a cysteine residue is important for successful crosslinking of galectin to its glycoprotein ligand using a photoactivatable sulfhydryl reagent

We have employed a combination of cysteine mutagenesis and chemical crosslinking using a photoactivatable sulfhydryl reagent, benzophenone-4-maleimide, to obtain a covalent complex between human galectin-1 and a model glycoprotein ligand, asialofetuin. We previously obtained a crosslinked product when Lys(28) of the cysteine-less form of human galectin-1 was mutated to cysteine. To investigate whether substituting either of the two flanking amino acid residues in the same β-strand, Ala(27) and Ser(29), to cysteine could result in crosslinking to the bound asialofetuin, two cysteine-containing mutants were generated. Although both the mutants adsorbed to asialofetuin-agarose and were eluted with 0.1 M lactose, confirming their ability to interact with asialofetuin, these mutants did not crosslink to the bound glycoprotein ligand following treatment with benzophenone-4-maleimide. Therefore the orientation of the side chain of the introduced cysteine residue apparently plays an important role in the crosslinking reaction.     

Depolarization potentiates TRAIL-induced apoptosis in human melanoma cells: role for ATP-sensitive K+ channels and endoplasmic reticulum stress

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is promising for cancer treatment owing to its selective cytotoxicity against malignant cells. However, some cancer cell types, including malignant melanoma cells, are resistant to TRAIL-induced apoptosis. Therefore, drugs that can amplify TRAIL cytotoxicity are urgently required. Depolarization of the plasma membrane potential is associated with apoptosis induced by a variety of death-inducing agents but its role in apoptosis remains a matter of debate. We found that TRAIL treatment resulted in robust depolarization in human melanoma cells with a considerable lag?(2-4?h). Moreover, membrane-depolarizing agents, including K+ and ATP-sensitive K+ (KATP) channel inhibitors glibenclamide and U37883A enhanced TRAIL-induced apoptosis. On the contrary, inhibitors of calcium- and voltage-dependent K+ channels and mitochondrial KATP channels had no such effects. Melanocytes were insensitive to TRAIL-induced depolarization and apoptosis as well as to the sensitization by membrane-depolarizing agents despite their substantial surface expression of death receptors. TRAIL induced robust activation of X-box-binding protein-1 and caspase-12, both of which were enhanced by the K+ and KATP channel inhibitors, but not by other K+ channel inhibitors. Finally, caspase-12-selective inhibitor completely abolished the amplification of apoptosis. These findings suggest that depolarization promotes endoplasmic reticulum stress-mediated death pathway, thereby amplifying TRAIL cytotoxicity. Thus, membrane-depolarizing agents such as KATP channel inhibitors may have therapeutic potential in the treatment of TRAIL-resistant cancer cells without impairing tumor-selectivity.