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.

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).

Anticancer efficacy in vivo and in vitro, synergy with 5-fluorouracil, and safety of recombinant methioninase.

The elevated exogenous-methionine dependency of tumors for growth has been observed in all major cancer cell types. We have previously cloned a methioninase (rMETase) from Pseudomonas putida to deplete methionine. Growth inhibition followed by apoptotic cell death was induced by treatment of tumor cells with rMETase in vitro. A single i.p. injection of 300 units of rMETase can lower the serum methionine level in the mice from 70 microM to less than 1 microM within 2 h and maintain this depleted level for 8 h. Repeated dosing of rMETase of tumor-bearing mice could be administered without acute immune-hypersensitivity. rMETase treatment demonstrated growth inhibitory activity against human tumors in nude mice, including those which were multiple drug-resistant. No body weight loss or hematotoxicity, except a slight anemia, was found throughout the therapy. The combined treatment of the Lewis lung carcinoma with a fixed rMETase dose and increasing doses of 5-fluorouracil (5-FU) resulted in a dose-dependent enhanced antitumor efficacy for survival as well as tumor growth inhibition. Thus, methionine depletion by rMETase potentiates the antitumor efficacy of 5-FU. The data presented in this report thus indicate that rMETase is active alone, is synergistic in combination with 5-FU, and has negligible toxicity suggesting a novel clinical approach for effective cancer therapy.

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.

Sequence-dependent antitumour efficacy of combination chemotherapy of nedaplatin, a novel platinum complex, with 5-fluorouracil in an in vivo murine tumour model.

The antitumour efficacy of a sequential combination of nedaplatin (NDP) and 5-fluorouracil (5-FU) was evaluated using Lewis lung carcinoma in vivo. NDP was developed as a second generation platinum complex. Because it has greater antitumour activity and lower nephrotoxicity than cisplatin (CDDP), we also compared the antitumour activity of NDP plus 5-FU with that of CDDP plus 5-FU. A fixed 5-FU dose was injected daily for 5 days and increasing doses of either NDP or CDDP were injected once via the tail vein into the Lewis lung carcinoma-implanted mice. The sequential administration of either NDP or CDDP prior to 5-FU (NF or CF therapy) resulted in severe body weight loss followed by the death of the tumour-bearing mice when the high-dose of NDP or CDDP was administered. In contrast, the sequential administration of 5-FU prior to NDP or CDDP (FN or FC therapy) resulted in synergistically enhanced inhibition of tumour growth and prolonged survival in comparison with NDP, CDDP or 5-FU monotherapy. At the high-dose of NDP in FN therapy, a reduction of tumour size and long-term tumour-free survival were frequently observed. The survival effect of the combinations of NDP with 5-FU was superior to those of the combination of CDDP with 5-FU. In conclusion, the sequence-dependent antitumour efficacy and toxicity of the combination of NDP or CDDP with 5-FU was demonstrated in this study, and FN therapy appeared to be the most efficient regimen as a clinical therapy.

Neutralizing monoclonal antibodies against recombinant human interleukin-2.

A series of hybridoma cell lines which produce monoclonal antibodies (MAbs) against recombinant human interleukin-2 (rIL-2) have been established by fusion of murine myeloma cell line P3-NS1-1-AG4-1 and spleen cells of BALB/c mice which had been immunized with rIL-2. 48 hybridoma strains were selected by a solid-phase screening method which produced MAbs reacting with IL-2: four MAbs, L-15, L-20, L-34, and L-61, exhibited strong inhibition of the proliferating effect of rIL-2 on IL-2-dependent cell lines, NK7 and CTLL-2. L-61, the most potent MAb among them, also neutralized natural human IL-2, while the other three MAbs were unreactive. All the four MAbs were specific to human IL-2: they did not cross-react with mouse or rat IL-2. These MAbs are expected to be useful tools in the investigation of IL-2 function.

Differential effect of cyclosporine in vivo on the distribution of T cell subsets in the thymus, spleen, and lymph nodes.

Cyclosporine treatment of BALB/c mice (at a dose of 20 mg/kg every other day for 3 weeks) caused a remarkable reduction in the PNA-, L3T4+Lyt-2- subset of thymocytes. A significant reduction of the L3T4+Lyt-2-subset was also observed in both the lymph node and spleen cells of CsA-treated mice, though the degree of the reduction was lower than that in thymocytes. Both lymph node and spleen cells from CsA-treated mice showed a significant increase in the percentage of Thy-1.2 negative, L3T4-Lyt-2- cells (perhaps B cells). Thymocytes from CsA-treated mice showed a reduction in the in vitro proliferative responses to Con A and PHA. On the other hand, there was a slight, but not significant, decrease in the responses of lymph node cells to Con A, PHA and LPS. Spleen cells from CsA-treated mice showed a significant reduction in the responses to Con A and PHA, though the degree of the reduction was lower than that of thymocytes. There was a significant decrease in the proliferative response of spleen cells to LPS. These results suggest that CsA affects both thymus and spleen cells in vivo, preferentially impairing the L3T4+Lyt-2- subset (helper T cells or their precursors) within the thymus. The lymph node cells seem to be relatively spared from the in vivo effect of CsA compared with cells in the thymus and spleen.

Antigen-presenting capacity of guinea pig B lymphocytes in T cell proliferative response in vitro.

The capacity of normal (unprimed) B cells and keyhole-limpet-hemocyanin(KLH)-primed B cells to present the antigen KLH to KLH-primed T cells in the guinea pig was examined with in vitro assay of lymphocyte proliferative response. Antigen-pulsed B-lymphocyte population, extensively devoid of macrophages (M phi), induced the proliferative response of KLH-primed T lymphocytes, although less effectively than the peritoneal M phi. The antigen presentation was antigen-specific. There was no substantial difference between the magnitude of T-cell response induced by KLH-primed B-cell population and that stimulated by unprimed, normal B-cell population. The antigen-presenting capacity of the B-cell population was abrogated by pretreatment with anti-guinea pig immunoglobulin (Ig) or anti-I-region-associated (Ia) antigen antiserum and complement, whereas it was not affected with anti-guinea pig M phi antiserum and complement. From studies using strain 2 and strain 13 guinea pigs, histocompatibility requirement between antigen-pulsed B cells and antigen-reactive T cells was suggested: B lymphocytes, as well as M phi, did elicit proliferative response to specific antigen, KLH or purified protein derivative of tuberculin (PPD), in syngeneic, but not in allogeneic, T cells. These results suggest that the Ia-positive, surface-Ig-bearing guinea-pig B lymphocytes - not only KLH-primed B cells but also unprimed B cells - are capable of presenting antigen to primed T cells in a major histocompatibility complex(MHC)-restricted fashion.

A new murine model of autoimmune orchitis induced by immunization with viable syngeneic testicular germ cells alone. II. Immunohistochemical findings of fully-developed inflammatory lesion.

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by two or three subcutaneous injections of viable syngeneic testicular germ cells, without the use of any adjuvants or immunopotentiators and that the disease induced was characterized by a complete lack of epididymitis despite a definite orchitis with hypospermatogenesis. In this report, immunohistochemical characterization of immune cells in the fully-developed orchitic lesion was carried out using monoclonal antibodies and immunoperoxidase staining. Thy-1.2+ cells, Mac-1+ cells, B220+ cells and cytoplasmic Ig-bearing cells in the lesion were estimated to be approximately 30, 15, 20 and 30% of all inflammatory cells, respectively. Major phenotype of T cells in the lesion was CD4+ (approximately 85%) with the remainder (approximately 15%) being CD8+. The percentages of cytoplasmic IgG-, IgA- and IgM-bearing cells were estimated as approximately 35, 60 and 5% of all cytoplasmic Ig-bearing cells, respectively. Deposits of immunoglobulins and third component of complement were identified on the basement membrane of the seminiferous tubules, interstitium between the tubules, vessel endothelium and degenerated germ cells in the lesion. Circulating antibodies directed against the acrosomal portion of germ cells were detected in IgG and IgM classes but not in IgA class. Inflammatory cells (including macrophages, B cells and, probably, activated T cells) in the lesion were Ia+, but Leydig cells, Sertoli cells and germ cells did not stain for Ia at all.

Unusual histiocytic tumor of the small intestine.

A case of unusual sarcoma of the small intestine is described. Ultrastructural observation, enzyme histochemical studies, and functional analysis, such as phagocytic capacity and surface markers, were helpful in defining the neoplastic cells as histiocytes. Autopsy findings are presented.

Reaction of 20S proteasome: shift of SDS-dependent activation profile by divalent cations.

The multicatalytic endopeptidase complex (20S proteasome) is a latent high-molecular-mass multisubunit proteinase. In many investigations, SDS has been used as a proteasome activator at some fixed concentration that was apparently optimal. This study examined the effects of various divalent cations on the SDS-dependent peptidase and casein degradation activities of 20S proteasome purified from Xenopus laevis oocytes at a series of SDS concentrations and the correlation between these effects and the critical micelle concentration (CMC) of SDS. Surprisingly, it was found that divalent cations such as Mg2+ markedly shifted the SDS-dependent activation profiles to a lower concentration range. Ca2+, Mn2+, Co2+, and Zn2+ also markedly reduced the optimum SDS concentration in the Suc-Leu-Leu-Val-Tyr-MCA hydrolysis reaction: for example, 5 mM Co2+ reduced the optimum SDS concentration from 0.065 to 0.005%. However, in all cases examined the optimum concentrations were below the CMC. Cu2+, Hg2+, and Cd2+ strongly inhibited the SDS-dependent maximum activity without remarkably shifting the optimum SDS concentration. No correlation between the shift and the inhibition was recognized. Most interestingly, remarkable activation of casein degradation by SDS was observed only by addition of the divalent cations Mg2+, Ca2+, and Mn2+. These cations might be essential for casein degradation. The activation and inactivation ranges of SDS concentration varied with the species of substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

PKC and tyrosine kinase involvement in amyloid beta (25-35)-induced chemotaxis of microglia.

Microglia are activated by amyloid beta (Abeta) in vivo and in vitro, and Abeta-activated microglia may be involved in the pathogenesis of Alzheimer's disease (AD). We investigated the mechanism of microglial chemotaxis induced by Abeta (25-35), an active fragment of Abeta. Abeta (25-35) 0.1 and 1 nM stimulated microglial chemotaxis. The protein kinase C (PKC) inhibitors chelerythrine (0.5 and 2 microM), calphostin C (1 microM) and staurospine (10 nM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). The chemotactic effect of Abeta (25-35) on microglia was desensitized by pretreatment of microglia with 1 ng/ml 12-O-tetradecanoylphorbol 13-acetate (TPA). Pretreatment of cells with Abeta (25-35) (1 nM) also desensitized the chemotactic effect by Abeta (25-35) (1 nM). The desensitization by TPA or Abeta (25-35) was inhibited when staurosporine was present in the pretreatment media. The tyrosine kinase inhibitor herbimycin A (0.1 and 1 microM) significantly inhibited the microglial chemotaxis induced by Abeta (25-35) (1 nM). Based on these observations, it seems likely that PKC and tyrosine kinase are involved in the Abeta-induced chemotaxis of microglia.

Adjuvant hepatic intra-arterial chemotherapy after potentially curative hepatectomy for liver metastases from colorectal cancer: a pilot study.

The site most at risk of recurrence after hepatectomy is the remaining liver. Therefore a study was conducted of the use of hepatic intra-arterial and oral chemotherapy for colorectal metastases to prevent intrahepatic recurrence. Our regimen consisted of intra-arterial 5-fluorouracil (5-FU), mitomycin C (MMC) and oral 1-hexylcarbamoyl-5-fluorouracil (HCFU). Sixteen patients were treated. Median total dose of 5-FU was 8.1 g, MMC was 43 mg, and HCFU was 54 g, respectively. Median follow-up period was 21 months. The recurrence rate of the remaining liver was 31%. With respect to the number of hepatic metastases, there was no recurrence in patients with a single deposit. On the other hand, the intrahepatic recurrence rate of patients with multiple deposits was 45%. Of six patients with more than five hepatic deposits, however, only one patient had developed recurrent disease in the liver. Chemical sclerosing cholangitis (19%) was the most serious complication. Consequently, we propose that a prospective randomized trial should be carried out to establish the effects of this type of adjuvant chemotherapy to reduce possible recurrences in the remaining liver.

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.

Postoperative follow-up studies on cancer of the colon and rectum.

We followed up 790 patients with cancer of the colon and rectum treated at our hospital since 1962. The curative resection rate in the rectum and anus was 78 percent but decreased as the distance of the lesion from the anus increased. In the ascending colon the rate was 59.8 percent. The patients undergoing curative resection had a 5 year survival rate of 65 percent and a 10 year survival rate of 45 percent. The closer to the anus, the poorer the prognosis. Prognosis is greatly influenced by the stage at diagnosis. Surgical results have improved annually due to progress in diagnostic and therapeutic procedures. Factors greatly influencing prognosis were the presence of lymph node metastasis, the degree of invasion of the intestinal wall and the site of the primary lesion. Lymph node metastasis was an especially important prognostic factor. In cancer of the rectum primary recurrences were most often found in local sites (66 percent of cases), followed by metastasis to the liver and the lung. However, in cancer of the colon metastasis in both the liver and the peritoneum was observed in 67 percent of the cases. On the basis of these results, methods to prevent local recurrence of cancer of the rectum as well as metastasis to the liver from cancer of the colon must be developed. When extended abdominopelvic wide dissection was performed, the incidence of local recurrence decreased.

A safe method of intrahepatic biliary drainage in obstructive jaundice.

In malignant disease with severe obstructive jaundice, survival after operative intervention is extremely low. Simple external biliary drainage is desirable for these patients, as a preliminary procedure for radical operation or long-term palliation. Intrahepatic biliary drainage at the anterior edge of the liver can be performed easily and safely under local anesthesia in case of obstruction of the proximal bile ducts, just as cholecystostomy is indicated for obstruction of the common bile duct.

Lymphatic spread and its prognostic value in patients with rectal cancer.

The incidence of lymph node metastasis in 423 patients with resected rectal cancer was 51.4 percent. There was no correlation between the size of the tumor and metastasis when the maximum diameter of the tumor exceeded 3 cm, and metastasis was seen in more than 50 percent of the patients. Metastasis was seen in 17.9 percent of the patients with cancer limited to the mucosa (mucosal and submucosal layer), in 37.8 of those with penetration limited to the propria muscle, and in more than 50 percent of those with penetration to the serosa or invasion into other organs. With lower rectal cancer, lateral metastasis was seen in 23 percent of the patients with advanced cancer, and inguinal lymph node metastasis in 6 percent. The 5 year survival rate of Dukes' C patients was as low as 33 percent, but was 52.9 percent in the patients with metastasis to only the pararectal lymph node; the prognosis of lateral metastasis is poor. As a subclassification of Dukes' C, levels 1, 2, 3, 4, and 5 are proposed on the basis of the extent of lymphatic spread.

Oxysterols found in opacified cornea of fish.

We describe a new concept implicating oxidized cholesterol derivatives and very long-chain fatty acids as possible factors in the development of corneal opacification after death. Corneal tissues, removed from both fresh and stale fish eyes, were examined for cholesterol derivatives and fatty acids after methanolysis of lipids. Cholesta-3,5-dien-7-one, cholest-4-en-3-one, hexacosenoic acid, and hexacosenoic acid were identified via gas chromatography/mass spectrometry in opacified corneas, but not in significant amounts in fresh ones. The present study confirmed the presence of lipid hydrolysis and a peroxidation process in the opacified cornea.

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.

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.