Prebiotic properties of epilactose.

We recently reported that cellobiose 2-epimerase from Ruminococcus albus effectively converted lactose to epilactose. In this study, we examined the biological effects of epilactose on intestinal microbiota, bile acid metabolism, and postadministrative plasma glucose by animal tests. Dietary supplementation with epilactose or fructooligosaccharide (4.5% each) increased cecal wall weight and cecal contents and decreased the pH of the cecal contents in Wistar-ST rats. The number of total anaerobes tended to be greater in rats fed epilactose and fructooligosaccharide than in those fed the control diet. Lactobacilli and bifidobacteria were more numerous in rats fed epilactose and fructooligosaccharide diets than in those fed the control diet. Analysis of clone libraries of 16S rRNA suggests that supplementation with epilactose did not induce the proliferation of harmful bacteria belonging to classes Clostridia or Bacteroidetes. Epilactose, as well as fructooligosaccharide, inhibited the conversion of primary bile acids to secondary bile acids, which are suggested to be promoters of colon cancer. In addition, oral administration of epilactose did not elevate the plasma glucose concentration in ddY mice. These results clearly indicate that epilactose is a promising prebiotic. We also showed that cellobiose 2-epimerase converted lactose in cow milk and a spray-dried ultrafiltrate of cheese whey to epilactose. Cellobiose 2-epimerase may increase the value of dairy products by changing lactose to epilactose possessing prebiotic properties.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era.

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90% of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8% were identified as Candida albicans and 42.1% as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5%), C. lusitaniae (5.2%), C. tropicalis (4.6%), C. parapsilosis (4.6%), C. glabrata (2.8%), C. kefyr (1.7%), C. guilliermondii (1.7%), C. intermedia (1.1%), C. norvegensis (0.5%), and Rhodotorula rubra (1.7%). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 micro g/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

Oral Candida flora from Brazilian human immunodeficiency virus-infected patients in the highly active antiretroviral therapy era

One of the main opportunistic fungal infections amongst immunocompromised individuals is oral candidosis, which has been found in up to 90 percent of human immunodeficiency virus (HIV)-infected patients. This study employed yeasts isolated from the saliva and oral cavities of 114 HIV-infected patients living in Campinas, São Paulo. Of the isolates, 57.8 percent were identified as Candida albicans and 42.1 percent as non-C. albicans. The latter isolates were subsequently identified as C. krusei (7.5 percent), C. lusitaniae (5.2 percent), C. tropicalis (4.6 percent), C. parapsilosis (4.6 percent), C. glabrata (2.8 percent), C. kefyr (1.7 percent), C. guilliermondii (1.7 percent), C. intermedia (1.1 percent), C. norvegensis (0.5 percent), and Rhodotorula rubra (1.7 percent). Susceptibility of the isolates to amphotericin B, fluconazole, miconazole, and itraconazole was also determined by a microdilution method adopted by the National Committee for Clinical Laboratory Standards. The isolates demonstrated various susceptibilities to the antifungal agents. In particular 29 C. albicans and 13 non-C. albicans isolates showed low susceptibility to FLCZ (> 64 æg/ml). This study revealed huge diversity of Candida species, in particular the increasing emergence of non-C. albicans associated with the oral flora of HIV-infected patients.

Baicalin is a major component of PC-SPES which inhibits the proliferation of human cancer cells via apoptosis and cell cycle arrest.

BACKGROUND: PC-SPES is an eight-herb mixture that was shown to have activity against prostate cancer. Recently, we isolated a major component (6% of the total ethanolic extract) known as baicalin from PC-SPES by high performance liquid chromatography (HPLC). METHODS: Baicalin was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of various cancer types (PC-3, DU145, LNCaP prostate cancer cell lines, MCF-7 breast cancer cell line, HL-60 myeloblastic leukemia cell line, and NB4 promyelocytic leukemia cell line). The ability of baicalin to induce apoptosis of cancer cells was examined by both staining with Annexin V and detection of cleavage of Poly (ADP-ribose) polymerase (PARP)(3). Western blot analysis examined the effect of baicalin on levels of p21(waf1) and p27(kip1) in those cells. Futhermore, induction of differentiation in HL-60 cells was measured by expression of CD11b. RESULTS: Baicalin inhibited the clonal proliferation of LNCaP and PC3 prostate cancer cell lines, and the HL-60 and NB4 myeloblastic/promyelocytic leukemia cell lines with a 50% inhibition (ED(50)) that ranged between 6.4 x 10(-6) to 12 x 10(-6) mol/L. Cell cycle analysis showed that baicalin (2 x 10(-5) mol/L, 4 days) caused a G(0)/G(1) and G(2)/M accumulation of LNCaP and HL-60 cells, respectively. Concomitantly, differentiation and apoptosis were induced in HL-60 cells, as measured by expression of CD11b antigen, staining with annexin V, and detection of cleavage of PARP. Moreover, baicalin enhanced the expression of the cyclin-dependent kinase inhibitor, p27(kip1) in LNCaP and HL-60 cells. CONCLUSIONS: Baicalin inhibited the proliferation of cancer cells via apoptosis and cell cycle arrest, in which p27(kip1) may play a role. Baicalin may be a novel, adjunctive therapy for selected malignancies including prostate cancer.

HIV-1 protease inhibitors decrease proliferation and induce differentiation of human myelocytic leukemia cells.

Inhibitors of the protease of human immunodeficiency virus type 1 (HIV-1) may inhibit cytoplasmic retinoic acid-binding proteins, cytochrome P450 isoforms, as well as P-glycoproteins. These features of the protease inhibitors might enhance the activity of retinoids. To explore this hypothesis, myeloid leukemia cells were cultured with all-trans retinoic acid (ATRA) either alone or in combination with the HIV-1 protease inhibitors indinavir, ritonavir, and saquinavir. Consistent with the hypothesis, the HIV-1 protease inhibitors enhanced the ability of ATRA to inhibit growth and induce differentiation of HL-60 and NB4 myeloid leukemia cells, as measured by expression of CD11b and CD66b cell surface antigens, as well as reduction of nitroblue tetrazolium. Growth of ATRA-resistant UF-1 cells was also inhibited when cultured with the combination of ATRA and indinavir. Moreover, indinavir enhanced the ability of ATRA to induce expression of the myeloid differentiation-related transcription factor C/EBPepsilon messenger RNA in NB4 cells by 9.5-fold. Taken together, the results show that HIV-1 protease inhibitors enhance the antiproliferative and differentiating effects of ATRA on myeloid leukemia cells. An HIV-1 protease inhibitor might be a useful adjuvant with ATRA for patients with acute promyelocytic leukemia and possibly retinoid-resistant cancers.

Acute effects of caffeine administration on the ultrastructure of the golden hamster parathyroid gland.

This study was conducted to evaluate the acute effects of caffeine on the ultrastructure of the parathyroid glands in golden hamsters. Caffeine was given orally at either 2.5 mg (low dose) or 10 mg (high dose) per 100 g body weight. Caffeine caused a dose dependent decrease of the serum calcium level 2 hours after administration. Transmission electron microscopy of the parathyroid gland revealed that the volume densities occupied by the Golgi complexes and rough endoplasmic reticulum (RER) were found significantly higher 2 hours after receiving high dose of caffeine. Statistical analysis revealed no significant differences regarding to the bone mineral content (BMC) and bone mineral density (BMD). It is considered that the synthesis of parathyroid hormone is stimulated following caffeine administration.

Effects of long-term treatment with caffeine on the ultrastructure of the golden hamster parathyroid gland and tibia.

The ultrastructure of the parathyroid gland and the SEM appearances of the tibia were studied in hamsters with and without administration of caffeine. Caffeine was treated orally each day at either 2.5 mg (low dose) or 10 mg (high dose) per 100 g body weight for a period of 17 or 32 days. Statistical analysis showed no significant differences among all groups examined regarding the serum calcium level. Transmission electron microscopy of the parathyroid gland revealed that the volume densities occupied by the mitochondria, Golgi complexes and rough endoplasmic reticulum of caffeine-treated groups were found significantly higher when compared with controls. The number of secretory granules observed close to the cell membrane per total amount of these granules revealed significant increase in all caffeine-treated animals. The bone mineral content (BMC) values were closely related to body weight. In the high dose caffeine-treated hamsters increment of the mean BMC and body weight values was significantly lower than those of the controls after 32 days. In the scanning electron microscopic studies of the tibia, no alteration in the morphometric parameters was demonstrated. It is considered that the synthesis and release of parathyroid hormone is stimulated following caffeine consumption. Our data suggest that although chronic administration of caffeine in the hamster may slightly increase bone turnover as evidenced by the BMC decrease, bone morphometry was not altered. Thus the osteoporotic changes were not proved in this study.

PC-SPES: a unique inhibitor of proliferation of prostate cancer cells in vitro and in vivo .

BACKGROUND: Management of prostate cancer that has spread beyond the capsule is a difficult problem. Innovative and nontoxic approaches to the disease are urgently required. Recently, a commercially available herbal mixture called PC-SPES showed potent antitumor activities on a variety of malignant cells in vitro. METHODS: PC-SPES was evaluated for its ability to inhibit clonal growth, and to induce cell cycle arrest of three human prostate cancer cell lines (LNCaP, PC-3, and DU 145). Western blot analysis examined the effect of PC-SPES on levels of p21(waf1), p27(kip1), Bcl-2, and E-cadherin in the three cell lines; and telomerase activity was examined by telomeric repeat amplification protocol (TRAP) assay. Furthermore, the effect of oral PC-SPES (250 mg/kg/day) on growth of PC-3 and DU 145 tumors present in male BNX nu/nu triple immunodeficient mice was studied. LNCaP cells were not analyzed in mice because they grow only with difficulty in these immunodeficient mice. RESULTS: PC-SPES markedly inhibited clonal growth of LNCaP, PC-3, and DU 145 prostate cancer cells, with a 50% inhibition (ED50) at approximately 2 microl/ml. Pulse-exposure studies showed that a 5-day pulse-exposure to PC-SPES (2 microl/ml) in liquid culture achieved a 50% inhibition of PC-3 clonal growth in soft agar, suggesting that the growth inhibition mediated by the extracts remained after removal of PC-SPES. Cell cycle analysis using the prostate cancer cell lines found that PC-SPES induced a significant increase in the number of cells in G0-G1 and G2/M, with a concomitant decrease in the number of cells in S phase. PC-SPES (2 microl/ml, 4 days) increased slightly the levels of p21(waf1) in the three cell lines, decreased by 40% the levels of Bcl-2 in PC-3, and the levels of p27(kip1) and E-cadherin and telomerase were unchanged in each of the lines. In vivo treatment with oral PC-SPES of male BNX mice having DU 145 tumors produced significant inhibition of their growth (P < 0.001), with no objective side effects including blood chemistries, weights, or autopsy analysis. The PC-SPES showed no statistical effect on the in vivo growth of PC-3 cells. CONCLUSIONS: PC-SPES inhibits clonal proliferation of human prostate cancer cells both in vitro and in vivo, using a murine model.

Novel antiallergic and antiinflammatory agents. Part II: Synthesis and pharmacology of TYB-2285 and its related compounds.

A series of m-bis(glycoloylamino)benzene derivatives was synthesized by treatment of the corresponding m-diaminobenzene derivatives with glycoloyl chloride derivatives in pyridine. Hydrolysis of acetyl compounds gave hydroxy derivatives, from which other acyl derivatives could be synthesized. These compounds were tested in the rat PCA (passive cutaneous anaphylaxis) assay by oral administration. Benzonitrile derivatives (4c, 5c, 6c, 4h, 5h) exhibited notable inhibition in this assay. Compounds 5c and 6c also showed remarkable inhibition of eosinophil adhesion to TNF- (tumor necrosis factor) alpha-treated HUVEC (human umbilical vein endothelial cells) in the range of 10(-8)-10(-5) M. Compound 5c is now under investigation in Japan as TYB-2285 (Figure 1) for asthma and atopic dermatitis in phase II clinical studies.

Polymerase chain reaction amplification of Asp f I and alkaline protease genes from fungus balls: clinical application in pulmonary aspergillosis.

Asp fI(18 kDa) and alkaline protease (33 kDa) are the 2 major antigens which are derived from Aspergillus (A.) fumigatus and have been implicated as possible virulence factors in the pathogenesis of Aspergillus-induced diseases. We attempted to detect fragments of genes encoding both proteins from fungus balls obtained at surgery or autopsy by polymerase chain reaction (PCR) amplification and then used PCR to test clinical samples. Frozen-stored fungus ball samples from a patient with acute myeloid leukemia complicated by Aspergillus pneumonia and from a patient with pulmonary aspergilloma were studied. We successfully amplified a 315 bp PCR product, the target sequence for Asp f I, and a 747 bp PCR product as a target sequence for alkaline protease (ALP) in both cases. In addition, 13 clinical samples including sputum specimens from patients with pulmonary aspergillosis were also examined. PCR analysis for the Asp f I (ALP) gene in clinical samples showed positive results in 5/10 (6/10) patients with pulmonary aspergilloma and in 3/3 (1/ 3) patients with invasive pulmonary aspergillosis. Culture data on A. fumigatus revealed positive results in 3/9 patients with pulmonary aspergilloma and in 2/3 patients with invasive pulmonary aspergillosis. This method can be used to recognize the involvement of A. fumigatus in various clinical settings where conventional culture results are not readily available.

Effect of Pinellia ternata tuber on the efferent activity of the gastric vagus nerve in the rat.

Effect of intraduodenal infusion with the hot aqueous extract of Pinellia ternata tuber on the efferent discharges in the gastric branch of the vagus nerve was observed in the anesthetized rat. The infusion of the extract in doses of 2-15O mg per animal (c.a. 300 g, b.wt.) resulted in a dose-related increase in efferent activity of the vagal gastric nerve. The enhancement of the nerve activity following administration of 150 mg of this substance lasted longer than 90 min. It was observed that the suppressive effect on vagal gastric activity due to apomorphine and copper sulfate was antagonized by prior administration of the extract. From these observations it is suggested that Pinellia tuber acts as a facilitatory agent on gastric function.

[Antitumor spectrum of TUT-7 on rat ascitic hepatoma].

The antitumor activity of TUT-7, a new anthracycline compound, was compared to that of adriamycin in the screening system with rat ascites hepatomas. A marked antitumor effect was observed in most tumor lines, and the activity was assumed to be better than adriamycin.

Comparison of antianginal activity of nicorandil, propranolol and diltiazem with reference to the antianginal mechanism.

Nicorandil was compared with placebo, propranolol and low and high doses of diltiazem therapy in 12 patients with chronic stable angina pectoris to elucidate its antianginal mechanism. A computer-assisted treadmill exercise test was performed after administration of either placebo, 30 mg of nicorandil, 40 mg of propranolol, or low-dose 60 and high-dose 120 mg of diltiazem. Exercise duration and time to the onset of ischemia were significantly increased after each drug administration and there was no significant difference in the percent increase in exercise duration between nicorandil (44 +/- 7%), propranolol (47 +/- 11%) and high-dose diltiazem (39 +/- 5%) compared with placebo. Nicorandil increased exercise duration in patients with 1-vessel disease more effectively (7.5 +/- 0.7 minutes, p less than 0.05) than either propranolol or low-dose diltiazem (6.7 +/- 0.7, 6.1 +/- 0.9 minutes, respectively). The decrease in blood pressure obtained with nicorandil was approximately the same as that with diltiazem. Nicorandil increased exercise duration associated with higher peak double product compared with low-dose diltiazem. In contrast, high-dose diltiazem increased exercise duration at the same double product as low-dose diltiazem. Propranolol increased exercise duration at a lower level of peak double product. Because our previous study demonstrated that low-dose diltiazem yielded a plasma concentration high enough to reduce coronary tone, it appears unlikely that nicorandil will reduce coronary tone further and subsequently increased coronary reserve. Therefore, left ventricular preload reduction may be the mechanism responsible for higher values of double product obtained with nicorandil.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of antihepatotoxic activity of wuweizisu C and gomisin A1.

The mechanism of inhibitory action of wuweizisu C and gomisin A in carbon tetrachloride (CCl (4))-induced liver damage was investigated by determining the effects of these substances on the steps of the series of events leading to liver lesion. Although wuweizisu C and gomisin A exerted no inhibition in CCl (3) radical formation, both lignans inhibited CCl (4)-, ADP/Fe (3+)- and ascorbate/Fe (2+)-induced lipid peroxidation, and wuweizisu C elicited stronger effects than gomisin A which is parallel with the results on antihepatotoxic effects in CCl (4)-induced cytotoxicity, indicating that anti-oxidative action plays an important part in the antihepatotoxic activity of wuweizisu C and gomisin A.

Antitumor activity of methanol extract from roots of Agrimonia pilosa Ledeb.

To evaluate the antitumor activity of Agrimonia pilosa LEDEB., the effects of the methanol extract from roots of the plant (AP-M) on several transplantable rodent tumors were investigated. AP-M significantly prolonged the life span of S180-, Meth-A fibrosarcoma- and MM-2 mammary carcinoma-bearing mice by intraperitoneal (i.p.) pre- or postmedication. AP-M also inhibited the growth of S-180 solid type tumor. On the other hand, the prolongation of life span induced by AP-M on S-180 ascites type tumor-bearing mice was markedly minimized or abolished by the pretreatment of cyclophosphamide. AP-M showed considerably strong cytotoxicity on MM-2 cells in vitro, but the effect was diminished to one-tenth by the addition of serum to the culture. Against the host animals, the peripheral white blood cells in mice were significantly increased from 2 to 5 days after the i.p. injection of AP-M. On 4th day after the injection of AP-M, the peritoneal exudate cells which possessed the cytotoxic activity on MM-2 cells in vitro were also increased to about 5-fold those in the non-treated control. The spleen of the mice was enlarged, and the spleen cells possessed the capacity to uptake 3H-thymidine. However, AP-M did not show direct migration activity like other mitogens against spleen cells from non-treated mice. These results indicate that the roots of Agrimonia pilosa contain some antitumor constituents, and possible mechanisms of the antitumor activity may be some host-mediated actions and direct cytotoxicity.

Reduced citrovorum factor rescue for high-dose methotrexate therapy in childhood malignancies.

Clinical toxicities and pharmacokinetics of methotrexate (MTX), associated with reduced citrovorum factor (CF) neutralization, were studied on 279 infusions in 25 children with various malignancies. MTX, at 1000-8400 mg/m2, was infused during six to 24 hours with multiple schedules of reduced CF rescue. Plasma MTX levels ranged from 7.0 X 10(-5) to 7.0 X 10(-4) M during MTX infusion. The levels declined rapidly with a two-phase elimination pattern (t1/2 = 1.2-2.5 hours, t1/2 = 18-32 hours). The folate level in the plasma ranged from 5 X 10(-7) M to 1.4 X 10(-6) M when CF was administered every six hours or every three hours, respectively. Limited bone marrow suppression was seen in only seven percent of infusions, with moderate elevation of GOT and GPT in 20% of infusions, and stomatitis in only 2.6% of infusions, despite reduction in the total dose of CF from 225 mg to 105 mg and despite delaying CF initiation from nine hours to thirty-six hours after the start of MTX infusion.