Antimicrobial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria.

AIMS: The aim of this study is to assess the antibacterial activity of sodium citrate against Streptococcus pneumoniae and several oral bacteria. METHODS AND RESULTS: The antibacterial activity was determined by broth microdilution method. The results showed that although Enterocuccus faecium OB7084 and Klebsiella pneumoniae OB7088 had high tolerance to sodium citrate, several oral bacteria including Fusobacterium nucleatum JCM8532(T) , Streptococcus mutans JCM5705(T) and Strep. pneumoniae NBRC102642(T) were susceptible. Furthermore, the bactericidal activity of sodium citrate against Strep. pneumoniae NBRC102642(T) was not influenced by pH in the range of 5·0-8·0, whereas that of sodium lactate was weakened at neutral or weak alkaline pH. When Strep. pneumoniae NBRC102642(T) was treated with sodium citrate for 2 h, many burst cells were observed. However, addition of MgCl(2) or CaCl(2) to an assay medium weakened the antimicrobial activity although ZnCl(2) or MnCl(2) did not influence. CONCLUSIONS: Independent of pH, sodium citrate inhibited the growth of oral bacteria, which suggests that the mechanism is different from that of sodium lactate. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this study would be available for understanding the antimicrobial property of sodium citrate.

Bacterial interactions in dental biofilm development.

Recent analyses with ribosomal RNA-based technologies have revealed the diversity of bacterial populations within dental biofilms, and have highlighted their important contributions to oral health and disease. Dental biofilms are exceedingly complex and multispecies ecosystems, where oral bacteria interact cooperatively or competitively with other members. Bacterial interactions that influence dental biofilm communities include various different mechanisms. During the early stage of biofilm formation, it is known that planktonic bacterial cells directly attach to surfaces of the oral cavity or indirectly bind to other bacterial cells that have already colonized. Adherence through co-aggregation may be critical for the temporary retention of bacteria on dental surfaces, and may facilitate eventual bacterial colonization. It is likely that metabolic communication, genetic exchange, production of inhibitory factors (e.g., bacteriocins, hydrogen peroxide, etc.), and quorum-sensing are pivotal regulatory factors that determine the bacterial composition and/or metabolism. Since each bacterium can easily access a neighboring bacterial cell and its metabolites, genetic exchanges and metabolic communication may occur frequently in dental biofilms. Quorum-sensing is defined as gene regulation in response to cell density, which influences various functions, e.g., virulence and bacteriocin production. In this review, we discuss these important interactions among oral bacteria within the dental biofilm communities.

Reduction of vitamin K concentration by salivary Bifidobacterium strains and their possible nutritional competition with Porphyromonas gingivalis.

AIMS: To assess the possibility that bifidobacteria compete with Porphyromonas gingivalis for their mutual growth factor vitamin K. This study also examined whether salivary Bifidobacterium species decrease vitamin K concentration in the growth medium. METHODS AND RESULTS: Sixty-five strains of Bifidobacterium were obtained from 20 of 24 periodontally healthy subjects. Bifidobacterium dentium was most frequently detected in the saliva of subjects, followed by Bifidobacterium adolescentis, Bifidobacterium longum, and Bifidobacterium urinalis. The growth of most Bifidobacterium isolates, except that of B. urinalis, was stimulated by vitamin K. Moreover, the isolates were capable of decreasing vitamin K after incubation, which suggests that bifidobacteria compete with P. gingivalis for vitamin K. In a co-culture, a representative strain -B. adolescentis S2-1 - inhibited the growth of P. gingivalis if it was inoculated in the medium before P. gingivalis. CONCLUSIONS: B. adolescentis S2-1 decreased vitamin K concentration and inhibited the growth of P. gingivalis by possibly competing for the growth factor. SIGNIFICANCE AND IMPACT OF THE STUDY: Salivary bifidobacteria may possess the potential to suppress the growth of P. gingivalis by reducing the growth factor(s) in the environment.

Quantitative measurement of tetrahydromenaquinone-9 in cheese fermented by propionibacteria.

Propionibacteria produce tetrahydromenaquinone-9 [MK-9 (4H)] as a major menaquinone (vitamin K2). This study aimed to determine the MK-9 (4H) concentration in commercial propionibacteria-fermented cheese. The MK-9 (4H) concentration was quantified using an HPLC instrument with a fluorescence detector after postcolumn reduction. Among the various cheese samples, the MK-9 (4H) concentration was highest in Norwegian Jarlsberg cheese, followed by Swiss Emmental cheese. In contrast, the MK-9 (4H) concentrations in Appenzeller or Gruyère cheeses were extremely low or undetected. Likewise, the concentrations in Comte and Raclette cheeses were lower than those in Jarlsberg and Emmental cheeses. In the present study, the MK- 9 (4H) concentration in cheese showed a correlation with the viable propionibacterial cell count and propionate concentration. This implies that the increase in propionibacteria contributed to the generation of MK-9 (4H) in cheese. We presumed, based on these results, that Swiss Emmental and Norwegian Jarlsberg cheeses contain a meaningful amount of vitamin K because of their high MK-9 (4H) concentrations (200 to 650 ng/g).

Amino acids and peptides. Part 39: a bivalent poly(ethylene glycol) hybrid containing an active site (RGD) and its synergistic site (PHSRN) of fibronectin.

Fibronectin contains the active sequence Arg-Gly-Asp (RGD), along with its synergic site Pro-His-Ser-Arg-Asn (PHSRN). However, the PHSRN peptide does not show synergic activity when it is mixed with the RGD peptide, indicating that a spatial array between RGD and PHSRN in fibronectin may be necessary for synergic activity. Here, we have used an amino acid type poly(ethylene glycol) derivative (aaPEG) to design a bivalent PEG hybrid of fibronectin active peptides. We prepared the aaPEG hybrid peptides PHSRN-aaPEG, aaPEG-RGD, and PHSRN-aaPEG-RGD, and tested their biological activity. Whereas aaPEG-RGD promoted cell spreading activity, PHSRN-aaPEG had no activity. The PHSRN-aaPEG-RGD hybrid strongly promoted cell spreading compared with aaPEG-RGD. These results suggest that the PHSRN sequence in the PHSRN-aaPEG-RGD molecule synergistically enhances the cell spreading activity of the RGD sequence, and that the bivalent aaPEG hybrid method may be useful for conjugating functionally active peptides.

Amino acids and peptides. XXXVIII. Facile synthesis of laminin-related peptide-poly(ethylene glycol) hybrids by the solid phase method.

Poly(ethylene glycol) (PEG) has been studied as a drug-carrier for proteins, but not for small peptides. Laminin, a cell adhesive protein, has Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence and peptides containing this sequence inhibit experimental metastasis. We have studied PEG hybrids of YIGSR and other small laminin-related peptides. In a previous paper, we reported preparation of YIGSR-PEG hybrids by combination of the solid phase method and the solution method, but the synthetic procedure was problematic. Here we report a facile synthesis of PEG hybrids of YIGSR (PEG-YIGSR, YIGSR-PEG, PEG-YIGSR-PEG) by the solid phase method.

Preclinical in vivo antitumor efficacy of nedaplatin with gemcitabine against human lung cancer.

The antitumor efficacy of the combination of nedaplatin (NDP) with gemcitabine (GEM) was evaluated. We also compared the antitumor activity of NDP plus GEM with that of cisplatin (CDDP) plus GEM or carboplatin (CBDCA) plus GEM. Ma44, which is a human lung cancer sensitive to GEM, and NCI-H460, which is a human lung cancer refractory to GEM, were used in this study. GEM was injected i.v. once followed by i.v. injection of NDP at an interval of approximately 30 min into tumor-bearing athymic mice. GEM was administered again 3 or 4 days thereafter. Combined dosing of NDP with GEM resulted in synergistically enhanced inhibition of tumor growth in the Ma44 tumor model. NDP plus GEM was also effective against Ma44 cells when given late in the therapy, a model for advanced disease. Potent augmentation of growth inhibition by NDP with GEM was also found with the NCI-H460 tumor model. The combination effect of NDP plus GEM appeared to be superior to that of CDDP plus GEM or CBDCA plus GEM in both tumor models. Toxicity in terms of blood cell numbers was not enhanced by the combination of NDP with GEM. These results suggest the effectiveness of combination of NDP with GEM for clinical therapy.

A new water-soluble N-protecting group, 2-(phenyl(methyl)sulfonio)ethyloxycarbonyl tetrafluoroborate, and its application to solid phase peptide synthesis in water.

A new water-soluble N-protecting group, 2-[phenyl(methyl)sulfoniolethyloxycarbonyl tetrafluoroborate, has been prepared and its application to solid phase peptide synthesis in water has been studied. Leu-enkephalin amide was successfully synthesized in water by the solid phase method using this protecting group.

Amino acids and peptides. XXXV. Facile preparation of p-nitroanilide analogs by the solid-phase method.

p-Nitroanilides of amino acids and peptides are widely used as the chromogenic substrates for the determination of the activity of proteolytic enzymes. However, the preparation of a p-nitroanilide is not easy, in part due to the low nucleophilicity of the amino group of p-nitroaniline. A facile preparation of p-nitroanilide analog by the solid-phase method was investigated. 5-Amino-2-nitrobenzoic acid (Anb5,2) was used instead of p-nitroaniline (pNA) for preparation of p-nitroanilide analogs. Anb5,2 was introduced on a p-methylbenzhydrylamine resin without protection of the amino group of Anb5,2 by the 2-(H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method in the presence of p-dimethylaminopyridine. The coupling reaction of a Nalpha-,NG-protected arginine with a Anb5,2-resin was difficult to achieve by common coupling methods (such as the carbodiimide and diphenylphosphoryl azide methods), but the phosphoryl chloride method was relatively successful. Synthetic benzoyl-Arg-Anb5,2-NH2 and benzoyl-Arg-pNA were hydrolyzed by trypsin and the both reaction mixtures exhibited same spectroscopic characteristics. H-D-Val-Leu-Arg-Anb5,2-NH2, an analog of human urine kallikrein substrate, was readily prepared by the solid-phase method. H-Arg-Anb5,2-OH and H-D-Val-Leu-Arg-Anb5,2-OH were also synthesized on a Wang resin by the solid-phase method. The aqueous solubility of these free-carboxyl materials was better than those of the corresponding amide analogs. 4-Amino-3-nitrobenzoic acid (Anb4,3) was also introduced on the p-methybenzhydrylamine resin, but the resulting H-Anb4,3-resin did not react with Nalpha,NG-protected arginine by any of the coupling methods.

Facile synthesis of a chitosan hybrid of a laminin-related peptide and its antimetastatic effect in mice.

Laminin, a cell adhesion protein, consists of three peptide chains (alpha-1, beta-1 and gamma-1). The beta-1 chain contains a Tyr-Ile-Gly-Ser-Arg (YIGSR) sequence that has been found to inhibit experimental metastasis in mice. We have prepared a hybrid of a water-soluble chitosan and a laminin-related peptide, and have examined its inhibitory effect on experimental metastasis in mice. A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-betaAla-OH (Ac-YIGSRbetaA-OH), was prepared by a solid-phase method. Ac-YIGSRbetaA-OH was then reacted with a water-soluble chitosan. BetaAla is a spacer and was placed to avoid racemization of the Arg residue when the peptide was coupled with chitosan. Although chitosan has amino groups, they did not react with the peptide. Four methods were tried to achieve a coupling reaction, the diphenylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotriazole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butyloxycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilitate its coupling with the peptide. After removal of the protecting group, the Gly-chitosan was coupled with Ac-YIGSRbetaA-OH by the water-soluble carbodiimide method to give Ac-YIGSRbetaAG-chitosan. Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, it actually potentiated the antimetastatic effect, demonstrating that chitosan may be effective as a drug carrier for peptides.

[Antiangiogenic and antitumor effect of BPHA, an orally-active matrix metalloproteinase inhibitor, in in vivo murine and human tumor model].

We examined the antiangiogenic and antitumor efficacy of a newly-developed matrix metalloproteinase (MMP) inhibitor, BPHA (N-biphenyl sulfonyl-phenylalanine hydroxiamic acid). BPHA potently inhibits MMP-2, 9 and 14 but not MMP-1, 3 or 7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMP tested. Daily oral administration of BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth and liver metastasis, whereas (-)BPHA did not. These results demonstrate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential without hematotoxic effect or loss of body weight.

Suppression of oncogenic transformation by hypothemycin associated with accelerated cyclin D1 degradation through ubiquitin-proteasome pathway.

Hypothemycin was originally isolated as an antifungal metabolite of Hypomyces trichothecoides. Here we report that treatment on v-K-ras-transformed NIH3T3 cells (DT cells) with hypothemycin caused drastic decrease in amount of cyclin D1 protein with concomitant prolongation of G1 phase in their cell cycle. Analysis using hypothemycin-resistant mutant of Schizosaccharomyces pombe (S. pombe) was carried out to show that S. pombe rhp6+ (homologue of Saccharomyces cerevisiae RAD6) and mammalian ubiquitin-conjugating enzyme 2 (ubc2) are the targets of hypothemycin or its downstream molecules in ubiquitin-conjugation process. Furthermore, in the presence of lactacystin, a specific inhibitor for proteasome, hypothemycin greatly enhanced the accumulation of multi-ubiquitinated form of cyclin D1 in DT cells. Therefore, it is indicated that hypothemycin facilitates ubiquitinating process of cyclin D1. In terms of malignant phenotype, hypothemycin inhibited anchorage-independent growth and reverted the morphology of DT cells. On the contrary, their morphology still remained transformed in the additional presence of lactacystin. Our results suggest that cyclin D1 is a key molecule working downstream in ras-signaling and that the transformation can be inhibited by the compound which can activate ubiquitin-proteasome pathway including degradation of cyclin D1.

Blood biochemical characteristics, cecal microbiota and short-chain fatty acid composition in fistula implanted rats.

We raised an experimental rat implanted with a cecal fistula and investigated various characteristics of fistula-implanted rats. Male F344/N Sic rats at 14 weeks of age were divided into three groups, the fistula group (n = 5) which consisted of fistula-implanted rats, the sham group (n = 7) which consisted of sham-operated rats, and the control group (n = 7) which were not subjected to any surgical procedure. Four weeks after the fistula implantation surgery, we compared the blood biochemical indices, the microflora composition and the short-chain fatty acids (SCFA) concentration in cecal contents of fistula-implanted rats with those of sham-operated and control rats. The blood albumin concentration of the fistula group was significantly lower than that of the sham group and the control group, and the hematocrit value of the fistula group was significantly lower than that of the control group, but there were no significant differences in the SCFA concentration and the microflora composition among these three groups. In conclusion, it was considered that the fistula-implanted rats are useful for taking cecal contents and determining the microflora composition and the metabolites concentration at any time, without disturbing the physiological functions of the intestinal tract.

Pre-operative chemoradiation therapy with 5-fluorouracil and low-dose daily cisplatin for esophageal cancer: a preliminary report.

BACKGROUND: A combination of chemotherapy and radiotherapy (chemoradiation therapy; CRT) has recently been developed to improve the survival of esophageal cancer patients. However, the optimal choice of chemotherapeutic agents and their doses, as well as chemotherapy and radiotherapy regimens, remain unclear. METHODS: Based on recent advances in knowledge on the radiosensitizing and biochemical modulation effects of chemotherapeutic agents, we have recently developed concurrent CRT which consisted of continuous 5-fluorouracil (5FU) administration (600 mg/m2/day, days 1-5) combined with a low dose of daily cisplatin administration (10 mg/m2/day, days 1-5, and 5 or 10 mg/m2/day, days 8-12 and 15-19) before each fraction of radiation (2 Gy each). To evaluate the efficacy and safety of our concurrent CRT, 10 esophageal cancer patients received one or one and a half courses of the CRT. RESULTS: All patients tolerated and completed a full course of the CRT. The effectiveness of the CRT on the primary tumor included pathologically or endoscopically complete responses in three patients (30%), partial response in five (50%), no response in two (20%) and tumoral downstaging (T-classification) in five (50%). Grade 2 and Grade 3 toxicity, seen in six patients, did not affect surgical operation. No patients showed CRT-related deaths. Eight patients (80%) underwent resection with no operative mortality. Of these, two patients (25%) showed pathologically or endoscopically complete responses, and four (50%) showed partial response. Three patients died of cancer after resection. The two inoperable patients showed a pathologically complete response and partial response, respectively. They were relieved of their cancer-related complaints and were living without hospitalization at the time of this analysis. CONCLUSIONS: These results suggest that the concurrent CRT based on the theoretical backgrounds is effective and has acceptable toxicities with maintaining its efficacy for the treatment of esophageal cancer patients.

Hereditary sensory neuropathy with deafness and dementia: a clinical and neuroimaging study.

We describe three sibling patients with autosomal dominantly inherited sensory neuropathy, sensorineural hearing loss and dementia. The features of cognitive-behavioral deficits in the patients, including executive dysfunction, apathy, indifference and inattention, were consistent with a frontal lobe dysfunction. Magnetic resonance imaging showed a diffuse brain atrophy. A fluorodeoxyglucose positron emission tomography in one patient and a single photon emission computed tomography in another demonstrated a glucose hypometabolism or a hypoperfusion in the medial frontal and thalamic regions. Primary frontal involvement or frontal dysfunction secondary to thalamic lesions may contribute to the nature of dementia in these patients.

Regioselective conjugation of chitosan with a laminin-related peptide, Tyr-Ile-Gly-Ser-Arg, and evaluation of its inhibitory effect on experimental cancer metastasis.

A conjugate from the YIGSR peptide and chitosan has been prepared on the basis of a regioselective modification strategy of chitosan, and its antimetastatic activity has been assayed. Chitosan was converted to its organosoluble derivative, 6-O-trityl-chitosan, in 3 steps, and then coupled with the peptide portion containing a spacer amino acid, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-OH [beta Ala; beta-alanine]. The product was treated with CHCl2CO2H to afford the desired conjugate, Ac-Tyr-Ile-Gly-Ser-Arg-beta Ala-chitosan, which proved to inhibit the experimental lung metastasis of B16BL6 melanoma cells in mice at lower doses than the parent peptide.

Correlation of antiangiogenic and antitumor efficacy of N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA), an orally-active, selective matrix metalloproteinase inhibitor.

The antiangiogenic activity and antitumor efficacy of a newly developed matrix metalloproteinase (MMP) inhibitor were examined. N-biphenyl sulfonyl-phenylalanine hydroxiamic acid (BPHA) potently inhibits MMP-2, -9, and -14, but not MMP-1, -3, or -7. In contrast, (-)BPHA, an enantiomer of BPHA, was inactive against all MMPs tested. Daily oral administration of 200 mg/kg BPHA, but not (-)BPHA in mice resulted in potent inhibition of tumor-induced angiogenesis, primary tumor growth, and liver metastasis. The growth inhibition activity of BPHA was 48% and 45% in a B16-BL6 melanoma and F2 hemangio-endothelioma model, respectively. BPHA also showed 42% inhibition of the liver metastasis of C-1H human colon carcinoma cells. These results indicate that selective MMP inhibition is correlated with antiangiogenic and antitumor efficacy and that the selective MMP inhibitor BPHA has therapeutic potential.

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.

A rare case of primary malignant mesothelioma originating from the rectovaginal tissue.

A 35 year-old female patient with pelvic malignant mesothelioma is described. The patient underwent total pelvic exenteration due to a pelvic tumor. Macroscopically, the resected tumor was located in the rectovaginal lesion with invasion into the rectal and vaginal wall, and around the internal urethral ostium. Light microscopically, the tumor predominantly consisted of sheets of plump round cells with acidophilic cytoplasm, and focally of tumor cells showing papillary growth pattern. The tumor cells showed remarkable cellular pleomorphism, and were both alcian blue and periodic acid-Schiff stain negative. Electron microscopically, these tumor cells had numerous long bush-like microvilli on their surface with increased length/width ratios. Positive staining with epithelial membrane antigen, cytokeratin, and vimentin, and negative staining with the carcinoembryonic antigen and S-100 protein were observed immunohistochemically. Based on these histological and immunohistochemical estimations, the tumor was diagnosed as a primary malignant mesothelioma originating from the rectovaginal tissue. Review of the literature confirmed the rarity of pelvic malignant mesothelioma. The possibilities of the pathogenesis of the tumor include the tumor's arising from the peritoneal remnant in the rectovaginal tissues, or from the epithelium of the secondary Mullerian system, which shares the same ancestry with the peritoneum.