Sample records for 131i-labeled iodo-azomycin galactopyranoside

  1. Iodine-124-labeled iodo-azomycin-galactoside imaging of tumor hypoxia in mice with serial microPET scanning.


    Zanzonico, Pat; O'Donoghue, Joseph; Chapman, J Donald; Schneider, Richard; Cai, Shangde; Larson, Steven; Wen, Bixiu; Chen, Yuchun; Finn, Ronald; Ruan, Shutian; Gerweck, Leo; Humm, John; Ling, Clifton


    Tumor hypoxia, present in many human cancers, can lead to resistance to radiation and chemotherapy, is associated with a more aggressive tumor phenotype and is an independent prognostic factor of clinical outcome. It is therefore important to identify and localize tumor hypoxia in cancer patients. In the current study, serial microPET imaging was used to evaluate iodine-124-labeled iodo-azomycin-galactoside ((124)I-IAZG) (4.2-day physical half-life) as a hypoxia imaging agent in 17 MCa breast tumors and six FSaII fibrosarcomas implanted in mice. For comparison, another promising hypoxic-cell PET radiotracer, fluorine-18-labeled fluoro-misonidazole ((18)F-FMISO), was also imaged in the same tumor-bearing animals. Twelve animals were also imaged with (18)F-labeled fluoro-deoxyglucose ((18)F-FDG). In addition, histological examination was performed, and direct measurement of tumor oxygenation status carried out with the Oxylite probe system. Two size groups were used, relatively well-oxygenated tumors in the range of 80-180 mg were designated as small, and those >300 mg and highly hypoxic, as large. Based on the data from 11 MCa and six FSaII tumors, both (124)I-IAZG and (18)F-FMISO images showed high tracer uptake in the large tumors. In (18)F-FMISO images at 1, 3-4, and 6-8 h post-injection (p.i.), there was considerable whole-body background activity. In contrast, (124)I-IAZG imaging was optimal when performed at 24-48 h p.i., when the whole-body background had dissipated considerably. As a result, the (124)I-IAZG images at 24-48 h p.i. had higher tumor to whole-body activity contrast than the (18)F-FMISO images at 3-6 h p.i. Region-of-interest analysis was performed as a function of time p.i. and indicated a tumor uptake of 5-10% (of total-body activity) for FMISO at 3-6 h p.i., and of ~17% for IAZG at 48 h p.i. This was corroborated by biodistribution data in that the tumor-to-normal tissue (T/N, normal tissues of blood, heart, lung, liver, spleen, kidney

  2. Tumor immunotherapy in the mouse with the use of 131I-labeled monoclonal antibodies

    SciTech Connect

    Zalcberg, J.R.; Thompson, C.H.; Lichtenstein, M.; McKenzie, I.F.


    This report describes the use of 131I-labeled monoclonal antibodies in two experimental models for tumor immunotherapy. In vitro treatment of the radiation-induced murine thymoma ITT-1-75NS with radiolabeled anti-Ly-2.1 significantly impaired subsequent tumor growth in vivo. However, in vivo treatment of this tumor, which previously had been injected into C57BL/6 mice, was unsuccessful. By contrast, in vitro treatment of a human colorectal tumor cell line (COLO 205) with 131I-labeled 250-30.6--a monoclonal antibody directed against a secretory component of normal and malignant gastrointestinal epithelium--completely inhibited subsequent tumor growth in BALB/c nude (nu/nu) mice. Furthermore, in vivo treatment of preexisting human colorectal tumor xenografts significantly impaired progressive tumor growth. Although some tumor inhibition was also produced by unlabeled 250-30.6 antibody, this response was considerably amplified by treatment with (131I)-labeled 250-30.6 (P less than .05), suggesting that in vivo treatment of human tumors with the use of 131I-labeled monoclonal antibodies may be clinically beneficial. The antithyroid drug propylthiouracil was used to reduce dehalogenation of the radiolabeled immunoglobulins in an attempt to improve their therapeutic efficacy.

  3. Radioimmunotherapy of human hepatocellular carcinoma xenografts with 131I-labelled antiferritin antibody.

    PubMed Central

    Saiful Alam, A. F.


    The effects of 131-labelled antiferritin polyclonal antibody for the treatment of established hepatocellular carcinoma (HC-04) in athymic nude mice were evaluated. 131I-labelled antiferritin antibody localised specifically to a subcutaneous tumour with a mean of 8.1% of the infused dose per gram of tumour at 24 h after infusion when the experiment was started 15 days after inoculation and with a mean of about 6.5% of the infused dose per gram of tumour when the experiment was started 30 days after tumour transplantation. The concentrations of 131I-antiferritin antibody in tumour delivered a mean of 1994 cGy to tumour following infusion of 500 microCi of radiolabelled antiferritin antibody in the early group and a mean of 1600 cGy in the late group. Treatment with 500 microCi led to regression of the tumour in 55% of animals in the early group and 44% in the late group. In contrast, unlabelled antiferritin and 131I-labelled IgG failed to exert any significant effect on tumour growth. The transplanted tumours in the early groups of animals had relatively higher concentration of ferritin than those in the late group. There was accelerated inhibition of tumour growth and prolonged survival in animals in the early group compared with those in the late group. PMID:2021533

  4. Radionuclide 131I-labeled multifunctional dendrimers for targeted SPECT imaging and radiotherapy of tumors

    NASA Astrophysics Data System (ADS)

    Zhu, Jingyi; Zhao, Lingzhou; Cheng, Yongjun; Xiong, Zhijuan; Tang, Yueqin; Shen, Mingwu; Zhao, Jinhua; Shi, Xiangyang


    We report the synthesis, characterization, and utilization of radioactive 131I-labeled multifunctional dendrimers for targeted single-photon emission computed tomography (SPECT) imaging and radiotherapy of tumors. In this study, amine-terminated poly(amidoamine) dendrimers of generation 5 (G5.NH2) were sequentially modified with 3-(4'-hydroxyphenyl)propionic acid-OSu (HPAO) and folic acid (FA) linked with polyethylene glycol (PEG), followed by acetylation modification of the dendrimer remaining surface amines and labeling of radioactive iodine-131 (131I). The generated multifunctional 131I-G5.NHAc-HPAO-PEG-FA dendrimers were characterized via different methods. We show that prior to 131I labeling, the G5.NHAc-HPAO-PEG-FA dendrimers conjugated with approximately 9.4 HPAO moieties per dendrimer are noncytotoxic at a concentration up to 20 μM and are able to target cancer cells overexpressing FA receptors (FAR), thanks to the modified FA ligands. In the presence of a phenol group, radioactive 131I is able to be efficiently labeled onto the dendrimer platform with good stability and high radiochemical purity, and render the platform with an ability for targeted SPECT imaging and radiotherapy of an FAR-overexpressing xenografted tumor model in vivo. The designed strategy to use the facile dendrimer nanotechnology may be extended to develop various radioactive theranostic nanoplatforms for targeted SPECT imaging and radiotherapy of different types of cancer.We report the synthesis, characterization, and utilization of radioactive 131I-labeled multifunctional dendrimers for targeted single-photon emission computed tomography (SPECT) imaging and radiotherapy of tumors. In this study, amine-terminated poly(amidoamine) dendrimers of generation 5 (G5.NH2) were sequentially modified with 3-(4'-hydroxyphenyl)propionic acid-OSu (HPAO) and folic acid (FA) linked with polyethylene glycol (PEG), followed by acetylation modification of the dendrimer remaining surface amines and

  5. Uptake and localization of /sup 131/I-labeled anti-calcitonin immunoglobulins in rat medullary thyroid carcinoma tissue

    SciTech Connect

    Gautvik, K.M.; Svindahl, K.; Skretting, A.; Stenberg, B.; Myhre, L.; Ekeland, A.; Johannesen, J.V.


    A medullary carcinoma of the thyroid gland (MCT) which has been transplanted repeatedly under the kidney capsule of Wag/Rij rats secretes calcitonin (CT) spontaneously. From 10--20 weeks after transplantation, immunoreactive serum calcitonin (iCT) is abnormally elevated and continues to rise parallel to tumor growth. The immunoglobulin fraction of the rabbit anti-CT antiserum raised against intact synthetic hormone, was purified and iodinated electrolytically. Specific activities of /sup 131/I-labeled immunoglobulin of 0.008--0.014 mCi/microgram protein were obtained with 80% preservation of CT binding activity. Wag/Rig rats with MCT tumor and increased serum iCT concentrations received intravenous injections of /sup 131/I-labeled immunoglobulins (0.054--0.811 mCi). The distribution of radioactivity in the rats was followed for 14 days using external scintigraphy in combination with radioactivity measurements of blood and different organs at the end of the observation period. The distribution of /sup 113/mIn was used as a marker for blood distribution. When the radioactivity ratios (/sup 131/I//sup 113/mIn) in tumor and different organs were related to that of blood which was set equal to unity, tumor tissue contained 3--6 times higher activity. Nonhyperimmune rabbit immunoglobulins or rabbit antirat prolactin immunoglobulins were not concentrated in MCT tissue, nor did anti-CT immunoglobulins localize in rat prolactin adenomas.

  6. Radionuclide (131)I labeled reduced graphene oxide for nuclear imaging guided combined radio- and photothermal therapy of cancer.


    Chen, Lei; Zhong, Xiaoyan; Yi, Xuan; Huang, Min; Ning, Ping; Liu, Teng; Ge, Cuicui; Chai, Zhifang; Liu, Zhuang; Yang, Kai


    Nano-graphene and its derivatives have attracted great attention in biomedicine, including their applications in cancer theranostics. In this work, we develop 131I labeled, polyethylene glycol (PEG) coated reduced nano-graphene oxide (RGO), obtaining 131I-RGO-PEG for nuclear imaging guided combined radiotherapy and photothermal therapy of cancer. Compared with free 131I, 131IRGO- PEG exhibits enhanced cellular uptake and thus improved radio-therapeutic efficacy against cancer cells. As revealed by gamma imaging, efficient tumor accumulation of 131I-RGO-PEG is observed after its intravenous injection. While RGO exhibits strong near-infrared (NIR) absorbance and could induce effective photothermal heating of tumor under NIR light irradiation, 131I is able to emit high-energy X-ray to induce cancer killing as the result of radio ionization effect. By utilizing the combined photothermal therapy and radiotherapy, both of which are delivered by a single agent 131IRGO- PEG, effective elimination of tumors is achieved in our animal tumor model experiments. Toxicology studies further indicate that 131I-RGO-PEG induces no appreciable toxicity to mice at the treatment dose. Our work demonstrates the great promise of combing nuclear medicine and photothermal therapy as a novel therapeutic strategy to realize synergistic efficacy in cancer treatment.

  7. Treatment of (131)I-labeled anti-CD147 monoclonal antibody in VX2 carcinoma-induced liver tumors.


    Niu, Huanzhang; Wang, Ruihua; Cheng, Jingliang; Gao, Shegan; Liu, Baoping


    Hepatocellular carcinoma (HCC) is a major health problem worldwide. CD147 has been reported to be overexpressed in HCC and blocking CD147 expression can decrease tumor growth. (131)I is often used in combination with other drugs to treat HCC and yields positive results. In this study, we combined the (131)I and CD147 monoclonal antibody to treat HCC in a rabbit VX2 animal model. In the (131)I-labeled CD147 antibody ((131)I-CD147-Ab) treatment group, the animals lived considerably longer than the animals in the other treatment groups. Metastasis and tumor growth in the (131)I-CD147-Ab treatment group were also inhibited. MMP2 and CD31 expression were significantly lower in the treatment group, whereas Tunel staining was overexpressed. These findings suggest that (131)I-CD147-Ab is a promising drug in the treatment of HCC, by inhibiting metastasis and growth and by decreasing the expression of MMP2 and CD31 or by inducing tumor necrosis. After testing the biochemical parameters, (131)I-CD147-Ab caused fewer side-effects in the animals.

  8. Therapy to target renal cell carcinoma using 131I-labeled B7-H3 monoclonal antibody

    PubMed Central

    Li, Xueqin; Zhang, Guangbo; Hou, Jianquan


    B7-H3 is a tumor-associated antigen that plays a critical role in potential tumor-targeted therapy. In this study, we aimed to assess the radiobiological effect of 131I-labeled B7-H3 monoclonal antibody (131I-4H7) in nude mice with human renal cell carcinoma (RCC) and evaluate the effect of 131I-4H7 on RCC treatment. The radiobiological activity and tumor uptake of 131I-4H7, and its effect on tumor growth were measured. 131I-4H7 was absorbed by the tumor and reached its maximal uptake rate (3.32% injected dose [ID]/g) at 24 h, at which point the drug concentration in the tumor was 7.36-, 2.06-, 1.80-, and 2.78-fold higher than that in muscle, kidneys, liver, and heart, respectively. Measurements and positron emission tomography–computed tomography imaging showed that tumor development was significantly inhibited by 131I-4H7. HE staining revealed that 131I-4H7 significantly injures tumor cells. Our results suggest that 131I-4H7 is markedly absorbed by the tumor and did suppress the development of RCC xenografted tumors in nude mice, which might provide a new candidate for antibody-mediated targeted radiotherapy in human RCC. PMID:27058890

  9. Pharmacokinetics of /sup 99m/Tc(Sn)- and /sup 131/I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments in nude mice

    SciTech Connect

    Zimmer, A.M.; Kazikiewicz, J.M.; Rosen, S.T.; Spies, S.M.


    The biodistribution, radioimmunoimaging, and high pressure liquid chromatography activity profiles of /sup 99m/Tc(Sn) and /sup 131/I-labeled anti-carcinoembryonic antigen monoclonal antibody fragments were compared. Nude mice, bearing specific (colon carcinoma, LS174T) and nonspecific (pancreatic carcinoma, MIA) xenografts were given injections of the respective radiolabeled antibody fragments and also of irrelevant /sup 125/I-labeled antibody fragments (MOPC-21). The animals were imaged at 24 h after being given injections, they were sacrificed, and biodistribution studies were performed. Results of the study showed high kidney uptake (48.6% injected dose (ID)/g +/- 8.1% (SD)) and low tumor uptake (1.5% ID/g +/- 0.6%) for /sup 99m/Tc(Sn)-labeled fragments and higher uptake (4.4% ID/g +/- 0.6%) for /sup 131/I-labeled fragments, resulting in a higher localization index for the radioiodinated monoclonal antibody fragments. Imaging results showed good tumor visualization at 24 h after injection for the /sup 131/I-labeled fragments and poor tumor visualization with predominant kidney uptake for /sup 99m/Tc(Sn)-labeled fragments. After radiolabeling, high pressure liquid chromatography analysis indicated that 131I was primarily associated with F(ab')2 fragments, whereas 99mTc was mostly associated with Fab' fragments.

  10. Quantitative uptake studies of /sup 131/I-labeled (E)-5-(2-iodovinyl)-2'-deoxyuridine in herpes simplex virus-infected cells in vitro

    SciTech Connect

    Gill, M.J.; Samuel, J.; Wiebe, L.I.; Knaus, E.E.; Tyrrell, D.L.


    We have synthesized a /sup 131/I-radiolabeled antiviral compound (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVdU) and shown that this agent was selectively trapped within rabbit kidney cells, infected in vitro by thymidine kinase-positive (TK+) herpes simplex virus (HSV). The uptake of /sup 131/I-labeled IVdU was specific, as it was not concentrated within either HSV (TK-) or mock-infected cells. In certain conditions, over 40% of the radiolabel was selectively trapped within HSV (TK+)-infected cells. This was a 20- to 30-fold increase over the uptake of /sup 131/I-labeled IVdU by HSV (TK-) or mock-infected cells. The uptake of /sup 131/I-labeled IVdU varied directly with (i) the dose of the virus used to infect the rabbit kidney cells; (ii) the concentration of radiolabeled IVdU added to the system; and (iii) the time of exposure of IVdU to infected cells. The ability of this agent to be trapped within HSV (TK+)-infected cells merits further evaluation in animal models as it has potential as a noninvasive, herpes-specific diagnostic test, in particular for HSV encephalitis.

  11. Transcatheter Arterial Chemoembolization Plus 131I-Labelled Metuximab versus Transcatheter Arterial Chemoembolization Alone in Intermediate/Advanced Stage Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis

    PubMed Central

    Zhu, Ze-xin; Liao, Ming-heng; Wang, Xiao-xue


    Objective The aim of the study was to compare transcatheter arterial chemoembolization (TACE) plus 131I-labelled metuximab with TACE alone for hepatocellular carcinoma (HCC). Materials and Methods A comprehensive search was conducted in PubMed, Embase, Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and Chinese BioMedical Literature Database with published date from the earliest to February 29th, 2016. No language restrictions were applied, but only prospective randomized controlled trials (RCTs) or non-RCTs were eligible for a full-text review. The primary outcome was the overall survival (OS) and effective rate (the rate of partial atrophy or complete clearance of the tumor lesion). The odds ratios (ORs) were combined using either the fixed-effects model or random-effects model. Results Eight trials (3 RCTs and 5 non-RCTs) were included, involving a total of 1121 patients. Patients receiving combined therapy of TACE plus 131I-labelled metuximab showed significant improvement in effective rate {OR = 4.00, (95% confidence interval [CI]: 2.40–6.66), p < 0.001}, 1-year OS (OR = 2.03 [95% CI: 1.55–2.67], p < 0.001) and 2-year OS (OR = 2.57 [95% CI: 1.41–4.66], p = 0.002]. Conclusion TACE plus 131I-labelled metuximab is more beneficial for treating advanced HCCs than TACE alone in terms of tumor response and OS. Large, multi-center, and blinded randomized trials are required to confirm these findings. PMID:27833404

  12. Radioimmunotherapy of human colon cancer xenografts by using {sup 131}I labeled-CAb{sub 1} F(ab'){sub 2}

    SciTech Connect

    Li Ling; Xu Huiyun; Mi Li; Bian Huijie; Qin Jun; Xiong Hua; Feng Qiang; Wen Ning; Tian Rong; Xu Liqing; Shen Xiaomei; Tang Hao; Chen Zhinan . E-mail:


    Purpose: Therapeutic efficacy, suitable dose, and administration times of {sup 131}I-CAb{sub 1} F(ab'){sub 2}, a new monoclonal antibody therapeutics specifically directed against a cell surface-associated glycoprotein of colon cancer, were investigated in this article. Methods and Materials: In human colon cancer xenografts, {sup 131}I-CAb{sub 1} F(ab'){sub 2} at the dose of 125 {mu}Ci, 375 {mu}Ci, and 1125 {mu}Ci were administrated intraperitoneally on Days 6 and 18 after implantation of HR8348 cells with CAb{sub 1} high reactivity. Survival time and tumor growth inhibition rate were used to evaluate the efficacy and safety of {sup 131}I-CAb{sub 1} F(ab'){sub 2} in treatment of colon cancer xenografts. Results: Treatment of 125, 375, and 1125 {mu}Ci {sup 131}I-CAb1 F(ab'){sub 2} did not significantly decrease the mean survival time of nude mice when compared with nontreated groups (p = 0.276, 0.865, 0.582, respectively). Moreover, the mean survival times of nude mice receiving 375 {mu}Ci and 1125 {mu}Ci {sup 131}I-CAb1 F(ab'){sub 2} were significantly longer than that of 5-FU-treated groups (p 0.018 and 0.042). Tumor growth inhibition rates of the first therapy were 35.67% and 41.37%, with corresponding {sup 131}I-labeled antibody dosage of 375 {mu}Ci and 1125 {mu}Ci. After single attack dosage, second reinforcement therapy may rise efficacy significantly. Tumor growth inhibition rates of 125 {mu}Ci, 375 {mu}Ci, and 1125 {mu}Ci {sup 131}I-labeled antibody on Day 20 posttherapy were 42.65%, 56.56%, and 84.41%, respectively. Histopathology examination revealed that tissue necrosis of various degrees was found in {sup 131}I-CAb1 F(ab'){sub 2}-treated groups. Conclusion: {sup 131}I-CAb{sub 1} F(ab'){sub 2} is safe and effective for colon cancer. It may be a novel and potentially adjuvant therapeutics for colon cancer.

  13. Kinetic study of internalization and degradation of sup 131 I-labeled follicle-stimulating hormone in mouse Sertoli cells and its relevance to other systems

    SciTech Connect

    Shimizu, A.; Kawashima, S. )


    The behavior of 131I-labeled follicle-stimulating hormone (FSH) after binding to cell-surface receptors in cultured Sertoli cells of C57BL/6NCrj mice was investigated. Sertoli cells cultured in F12/DME were pulse-labeled with 131I-FSH for 10 min at 4 degrees C, followed by cold chase for various periods of time. After the cold chase Sertoli cells were treated with 0.2 M acetate (pH 2.5) to dissociate membrane-bound 131I-FSH (surface radioactivity). The medium containing radioactivity after cold chase was mixed with 20% trichloroacetic acid, centrifuged, and the radioactivity of the supernatant was measured (degraded hormone). The radiolabeled materials associated with each process (surface binding, internalization, and degradation) were concentrated with ultrafiltration and characterized with gel filtration and/or thin layer chromatography. The effects of lysosomotropic agents, NH4Cl and chloroquine, were studied. The cold chase study at 32 degrees C showed that the surface radioactivity was the largest among the three kinds of radioactivities associated with each process immediately after pulse labeling, but the surface radioactivity rapidly decreased, while the internalized radioactivity increased. The cold chase study at 4 degrees C did not show such time-related changes in radioactivities, and a high level of surface radioactivity constantly persisted. The surface and internalized radioactivities were due to 131I-FSH, and the degraded radioactivity was mainly due to (131I)monoiodotyrosine. When Sertoli cells were cultured with lysosomotropic agents, the internalized radioactivity increased, while the degraded radioactivity decreased. Based on these observations, a kinetic model was proposed and the relationships among the surface, internalized, and degraded radioactivities and cold chase time were calculated algebraically.

  14. Immunohistology of carcinoembryonic antigen (CEA)-expressing tumors grafted in nude mice after radioimmunotherapy with 131I-labeled bivalent hapten and anti-CEA x antihapten bispecific antibody.


    Gautherot, E; Kraeber-Bodéré, F; Daniel, L; Fiche, M; Rouvier, E; Saï-Maurel, C; Thedrez, P; Chatal, J F; Barbet, J


    We have developed a pretargeting strategy, called the Affinity Enhancement System (AES), which uses bispecific antibodies (BsF(ab')2) to target radiolabeled bivalent haptens to tumor cells. We performed several radioimmunotherapy (RIT) experiments in nude mice grafted with LS174T colon carcinoma or TT medullary thyroid cancer. Mice were treated with 131I-labeled di-DTPA-indium-tyrosyl-lysine bivalent hapten (75-112 MBq) administered 15-48 h after anti-CEA x anti-DTPA-indium BsF(ab')2. Immunohistological studies were performed on tumors at their minimal relative volume (TT), on stabilized tumor nodules (LS174T), and on regrowing tumors (TT and LS174T). Untreated tumors were used as controls. On microscopic examination, regrowing tumors (2 months posttherapy) were similar to untreated tumors with cells showing their respective typical morphology (large cells with a high nucleocytoplasmic ratio for TT, small and very undifferentiated cells for LS174T). However, regrowing tumors showed larger necrotic areas and a higher mitotic index correlated with Ki-67 antigen staining. Immunostaining for CEA was as strong as for controls. By contrast, the immunohistology of TT tumors at their minimal relative volume (1 month posttherapy) or of LS174T residual nodules (8 months posttherapy) showed decreased mitotic indices correlated with poor Ki-67 antigen staining. Some clusters of LS174T presented with features of glandular lumen, which suggested a more differentiated and less aggressive status. In TT tumors, CEA expression remained unchanged (80-100% membrane and cytoplasmic staining), whereas only 70% of the LS174T tumors were stained, with 58% loss of the membrane expression. Repeated treatment early after the tumor has reached its minimal relative volume should thus be efficient and improve the overall efficacy of AES RIT.

  15. Preparation and properties of new co-crystals of ibandronate with gluco- or galactopyranoside derivatives.


    Oktabec, Zbynek; Kos, Jiri; Mandelova, Zuzana; Havelkova, Lenka; Pekarek, Tomas; Rezacova, Anna; Placek, Lukas; Tkadlecova, Marcela; Havlicek, Jaroslav; Dohnal, Jiri; Jampílek, Josef


    Mixtures of ibandronate monosodium salt with eleven gluco- and/or galacto-pyranoside derivatives as counterions were designed to prepare co-crystals with improved intestinal absorption. In general, gastrointestinal absorption of bisphosphonates after oral administration is approximately 1%. Co-crystals were generated by means of thermodynamically and/or kinetically controlled crystallization processes. Seventy-seven prepared samples were analyzed by means of FT-NIR, FT-Raman spectrometry and solid state NMR spectroscopy. New entities of ibandronate monosodium salt with phenyl-β-D-galactopyranoside were found and characterized. The absorption of these potential new co-crystals was investigated by means of PAMPA experiments. In the present study the relationships between the chemical structures of the studied compounds required for co-crystal generation are discussed.

  16. Value of the O-nitrophenyl-beta-D-galactopyranoside test to differentiate among the aerobic actinomycetes.

    PubMed Central

    Flores, M; Ford, E G; Janda, J M


    A comparative study to determine beta-D-galactosidase activity among 171 strains of aerobic actinomycetes (including mycobacteria and rhodococci) was performed by using two growth media and four O-nitrophenyl-beta-D-galactopyranoside (ONPG) substrates. The ONPG test was found to be a valuable screening test to differentiate between the ONPG-positive Nocardia spp. and the rapidly growing ONPG-negative mycobacteria and rhodococci. However, ONPG results varied significantly depending on the growth medium and test substrate used. PMID:2121796

  17. NOTE: Monte Carlo microdosimetry of 188Re- and 131I-labelled anti-CD20

    NASA Astrophysics Data System (ADS)

    Torres-García, E.; Garnica-Garza, H. M.; Ferro-Flores, G.


    The radiolabelled monoclonal antibody anti-CD20 has the property of binding to the CD20 antigen expressed on the cell surface of B-lymphocytes, thus making it a useful tool in the treatment of non-Hodgkin's lymphoma. In this work, the event-by-event Monte Carlo code NOREC is used to calculate the single-event distribution function f1(z) in the cell nucleus using the beta spectra of the 188Re and 131I radionuclides. The simulated geometry consists of two concentric spheres representing the nucleus and the cell surface embedded in a semi-infinite water medium. An isotropic point source was placed on the cell surface to simulate the binding of the anti-CD20 labelled with either 188Re or 131I. The simulations were carried out for two combinations of cell surface and nucleus radii. A method was devised that allows one to calculate the contribution of betas of energy greater than 1 MeV, which cannot be simulated by the NOREC code, to the single-event distribution function. It is shown that disregarding this contribution leads to an overestimation of the frequency-mean specific energy of the order of 9 12%. In general, the antibody radiolabelled with 131I produces single-event distribution functions that yield higher frequency-mean specific energies.

  18. Monte Carlo microdosimetry of 188Re- and 131I-labelled anti-CD20.


    Torres-García, E; Garnica-Garza, H M; Ferro-Flores, G


    The radiolabelled monoclonal antibody anti-CD20 has the property of binding to the CD20 antigen expressed on the cell surface of B-lymphocytes, thus making it a useful tool in the treatment of non-Hodgkin's lymphoma. In this work, the event-by-event Monte Carlo code NOREC is used to calculate the single-event distribution function f(1)(z) in the cell nucleus using the beta spectra of the (188)Re and (131)I radionuclides. The simulated geometry consists of two concentric spheres representing the nucleus and the cell surface embedded in a semi-infinite water medium. An isotropic point source was placed on the cell surface to simulate the binding of the anti-CD20 labelled with either (188)Re or (131)I. The simulations were carried out for two combinations of cell surface and nucleus radii. A method was devised that allows one to calculate the contribution of betas of energy greater than 1 MeV, which cannot be simulated by the NOREC code, to the single-event distribution function. It is shown that disregarding this contribution leads to an overestimation of the frequency-mean specific energy of the order of 9-12%. In general, the antibody radiolabelled with (131)I produces single-event distribution functions that yield higher frequency-mean specific energies.

  19. A rapid and sensitive fluorimetric β-galactosidase assay for coliform detection using chlorophenol red-β-D-galactopyranoside.


    Sicard, Clémence; Shek, Norman; White, Dawn; Bowers, Raymond J; Brown, R Stephen; Brennan, John D


    We report on a new fluorimetric assay for β-galactosidase (β-gal) and faecal coliform bacteria that utilizes a long-wavelength dye, chlorophenol red-β-D-galactopyranoside (CPRG), that has been widely used for colorimetric assays. The novel feature of this new assay is the unexpected development of a large fluorescence response from liberated chorophenol red (CPR) upon complexation with poly-L-arginine (pR) in solution. The binding of CPR to pR occurs through the sulphonate group of CPR, causing formation of a charge-transfer complex and up to a 70-fold increase in emission intensity. A major advantage of the assay is the ability to utilize excitation and emission wavelengths in the red end of the spectrum, which avoids common interferences obtained when using UV-absorbing dyes such as 4-methylumbelliferyl-β-D-galactopyranoside. We provide data on the utility of CPRG as a fluorimetric reporter for both β-gal and Escherichia coli ATCC 25922 and demonstrate optimized reaction conditions for rapid and sensitive detection of E. coli at a level of 1 colony-forming unit (cfu)/10 mL after 12 h of culture followed by a 1-h assay, which is below the regulatory limit for testing of recreational water.

  20. Umbelliferone β-D-galactopyranoside from Aegle marmelos (L.) corr. an ethnomedicinal plant with antidiabetic, antihyperlipidemic and antioxidative activity

    PubMed Central


    Background Aegle marmelos (L.) Corr. (Rutaceae), commonly known as bael, is used to treat fevers, abdomen pain, palpitation of the heart, urinary troubles, melancholia, anorexia, dyspepsia, diabetes and diarrhea in Indian traditional systems of medicine. The object of the present study was to evaluate the antidiabetic, antihyperlipidemic and antioxidant oxidative stress of umbelliferone β-D-galactopyranoside (UFG) from stem bark of Aegle marmelos Correa. in STZ (streptozotocin) induced diabetic rat. Methods Diabetes was induced in rat by single intraperitoneal injection of STZ (60 mg/kg). The rat was divided into the following groups; I – normal control, II – diabetic control, III – UFG (10 mg/kg), IV – UFG (20 mg/kg), V – UFG (40 mg/kg), VI – Glibenclamide (10 mg/kg, p.o., once a daily dose). Diabetes was measured by change the level blood glucose, plasma insulin and the oxidative stress were assessed in the liver by estimation of the level of antioxidant markers i.e. superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and Malondialdehyde (MDA) and antihyperlipidemic effect was measured by estimation of total cholesterol, triglycerides, LDL (low density lipoprotein) cholesterol, HDL (high density lipoprotein) cholesterol, VLDL (very low density lipoprotein) cholesterol. However in a study, the increased body weight was observed and utilization of glucose was in the oral glucose tolerance test. Result Daily oral administration of different dose of UFG for 28 days showed significantly (P < 0.001) decreased in fasting blood glucose level and improve plasma insulin level as compared to the diabetic control group. Also it significantly (P < 0.001) decreased the level of glycated hemoglobin, glucose-6-phosphatase, fructose-1-6-biphosphate and increased the level of hexokinase. UFG treatment decreased liver MDA and increased the level of SOD, GPx and CAT. UFG treatment of lipids it’s increased the level of cholesterol

  1. Synthesis of radioiodine-labeled 2-phenylethyl 1-thio-beta-D-galactopyranoside for imaging of LacZ gene expression.


    Choi, Joon Hun; Choe, Yearn Seong; Lee, Kyung-Han; Choi, Yong; Kim, Sang Eun; Kim, Byung-Tae


    A potent inhibitor of beta-galactosidase (EC, 2-phenylethyl 1-thio-beta-D-galactopyranoside (PETG), was radioiodinated for noninvasive imaging of LacZ gene expression. In order to introduce radioiodine to the phenyl ring of PETG, 2-(4-bromophenyl)ethanethiol was prepared and attached to the C-1 position of beta-D-galactose pentaacetate under conditions that resulted in the exclusive formation of the beta anomer. The bromo group of PETG was converted to the tributylstannyl group where radioiododemetallation was carried out. Radioiodine-labeled PETG tetraacetate was purified by HPLC, which can be used as a prodrug for biological evaluation or hydrolyzed to 2-(4-[123I/125I]iodophenyl)ethyl 1-thio-beta-D-galactopyranoside ([123I/125I]7) under basic conditions. The resulting radioiodine-labeled PETG was obtained in overall 62% radiochemical yield (decay-corrected) and with specific activity of 46-74 GBq/micromol.

  2. Synthetic mucin fragments: synthesis of O-sulfo and O-methyl derivatives of allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-alpha-D- galactopyranoside as potential compounds for sulfotransferases.


    Jain, R K; Piskorz, C F; Matta, K L


    Allyl 2-acetamido-4,6-O-(4-methoxybenzylidene)-2-deoxy-alpha-D-galact opy ranoside (1) was condensed with either 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (2) or 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide (14) in the presence of mercuric cyanide. Selective substitution with methyl, sulfo or both at desired positions, followed by the removal of protecting groups, afforded allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-6-O-methyl-alpha -D- galactopyranoside (5), allyl O-(6-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy-6- O-methyl-alpha-D-galactopyranoside (10), allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-6-O-sulfo-alpha- D- galactopyranoside sodium salt (13), allyl O-(6-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy- alpha-D-galactopyranoside (17) and allyl O-(3-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy- alpha-D-galactopyranoside (22). The structures of compounds 5, 10, 13, 17 and 22 were established by 13C NMR and FAB mass spectroscopy.

  3. Design, synthesis and enzymatic evaluation of 3-O-substituted aryl β-D-galactopyranosides as inhibitors of Trypanosoma cruzi trans-sialidase.


    Silva, Bruno L; S Filho, José D; Andrade, Peterson; Carvalho, Ivone; Alves, Ricardo J


    The trans-sialidase of Trypanosoma cruzi (TcTS) is a surface enzyme that modifies the parasite glycocalyx covering it with sialic acid. This process is essential to adhesion and invasion mechanisms in life cycle of the protozoan in the human host, making TcTS a very attractive molecular target for drug design. Using the TcTS substrate 3'-sialyllactose as prototype, D-galactose-derived potential inhibitors of TcTS were designed using strategies of molecular modification. Ten new aryl galactosides modified at carbon-3 were synthesized employing classical carbohydrate chemistry and dibutyltin oxide method for regioselective 3-O-alkylations and evaluated against TcTS by spectrofluorimetry. The 4-methoxycarbonyl-2-nitrophenyl 3-O-carboxymethyl-β-D-galactopyranoside was the most active compound inhibiting 21% of TcTS enzymatic activity at 1 mM.

  4. Factors affecting the relationship between the red marrow dose and myelotoxicity in patients receiving radioimmunotherapy with {sup 131}I-labeled anti-CEA monoclonal antibodies

    SciTech Connect

    Juweid, M.; Behr, T.M.; Sharkey, R.M.


    This study examined the relationship between the red marrow dose (RMD) and myelotoxicity in patients with CEA-producing tumors who received radioimmunotherapy (RAIT) with {sup 131}I-NP-4 and MN-14 anti-CEA MAbs. Eligibility criteria included no chemotherapy in the last 4 weeks prior to RAIT, no X-irradiation (XT) to >25% of marrow, WBC >3,000, platelets > 100,000, and Hg > 10.0. The RMD was estimated based on blood by assuming a red marrow-to-blood activity concentration ratio of 1.0. Myelotoxicity was evaluated based on standard RTOG criteria. Leukopenia or thrombocytopenia {ge} grade 3 was considered dose-limiting toxicity (DLT). A total of 109 eligible patients were assessed for myelotoxicity. Overall, reversible DLT occurred in 0/14 (0%), 1/25 (4%), 4/26 (15%), 6/25 (24%), 5/9 (55%), 3/6 (50%), and 3/4 (75%) patients receiving a mean RMD of 75, 150, 250, 350, 450, 550, and 650 cGy, respectively. Patients were further stratified into those who had chemotherapy (CHT) in the last 1-6 months prior to RAIT, and/or had XT or tumor metastases to 11-25% of their marrow (group 1), and those who had no CHT in the last 6 months and/or XT or metastases to {ge} 10% of the marrow (group 2). At 250, 350, 450, and 550 cGy, the incidence of DLT in group 1 was 4/17 (23%), 5/11 (45%), 4/4 (100%), and 3/3 (100%), respectively, compared to 0/9 (0%), 1/14 (7%), 1/5 (20%), and 0/3 (0%) in group 2. In conclusion, these data indicate that recent CHT, XT, and marrow metastases are important factors determining myelotoxicity after RAIT. Further, the relatively low incidence of myelotoxicity in group 2 (1/8, 12/5%) DLT at a RMD of 450-550cGy suggests that these patients may be able to tolerate an almost 2-fold higher dose than those in group 1. Although further refinement in identifying risk-factors for myelotoxicity are necessary, these results provide important insights for future planning of phase II trials.

  5. Synthesis of divalent ligands of β-thio- and β-N-galactopyranosides and related lactosides and their evaluation as substrates and inhibitors of Trypanosoma cruzi trans-sialidase

    PubMed Central

    Cagnoni, Alejandro J; Tesoriero, María Florencia; Kovensky, José


    Summary In this work we describe the synthesis of mono- and divalent β-N- and β-S-galactopyranosides and related lactosides built on sugar scaffolds and their evaluation as substrates and inhibitors of the Trypanosoma cruzi trans-sialidase (TcTS). This enzyme catalyzes the transfer of sialic acid from an oligosaccharidic donor in the host, to parasite βGalp terminal units and it has been demonstrated that it plays an important role in the infection. Herein, the enzyme was also tested as a tool for the chemoenzymatic synthesis of sialic acid containing glycoclusters. The transfer reaction of sialic acid was performed using a recombinant TcTS and 3’-sialyllactose as sialic acid donor, in the presence of the acceptor having βGalp non reducing ends. The products were analyzed by high performance anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). The ability of the different S-linked and N-linked glycosides to inhibit the sialic acid transfer reaction from 3’-sialyllactose to the natural substrate N-acetyllactosamine, was also studied. Most of the substrates behaved as good acceptors and moderate competitive inhibitors. A di-N-lactoside showed to be the strongest competitive inhibitor among the compounds tested (70% inhibition at equimolar concentration). The usefulness of the enzymatic trans-sialylation for the preparation of sialylated ligands was assessed by performing a preparative sialylation of a divalent substrate, which afforded the monosialylated compound as main product, together with the disialylated glycocluster. PMID:25670976

  6. Syntheses of the 3- and 4-thio analogues of 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-gluco- and galactopyranoside.


    Chen, Hong-Ming; Withers, Stephen G


    The syntheses of 4-nitrophenyl beta-glycosides of the 3-thio and 4-thio analogues of the two principal 2-acetamido-2-deoxy-hexoses found in living systems, GlcNAc and GalNAc, are described. While synthesis of the 4-thio analogues could be achieved via nucleophilic displacements of sulfonate derivatives with thioacetate, problems with neighbouring group acetamido participation necessitated the use of sulfamidate intermediates for the 3-thio analogues. These 3- and 4-thio analogues are employed in the chemo-enzymatic synthesis of thio-oligosaccharide analogues of structures present in glycosaminoglycans, glycoproteins and glycolipids.

  7. Phase I-II study of isotopic immunoglobulin therapy for primary liver cancer

    SciTech Connect

    Ettinger, D.S.; Order, S.E.; Wharam, M.D.; Parker, M.K.; Klein, J.L.; Leichner, P.K.


    A phase I-II study of isotopic immunoglobulin therapy was performed in 18 patients with primary liver cancer; 14 were evaluable for toxicity. The patients received a dose of 37-157 millicuries of 131I-labeled antibody. The dose-limiting factor appears to be hematologic toxicity, especially thrombocytopenia. An objective antitumor effect was seen in six of nine patients who were evaluable for response. Present results suggest that further clinical studies with isotopic immunoglobulin are indicated.

  8. Decreased thyroidal response to thyrotropin in diabetic mice

    SciTech Connect

    Bagchi, N.; Brown, T.R.; Shivers, B.; Lucas, S.; Mack, R.E.


    The effect of diabetes mellitus on the synthesis and secretion of thyroid hormone ws investigated in mice with streptozotocin-induced diabetes. Thyroid glands were labeled in vivo with 131I for 2 h. In control animals, TSH stimulated the synthesis of PB127I and 131I-labeled iodothyronines and simultaneously decreased the proportion of 131I-. These effects of TSH were not observed in diabetic animals but were demonstrable in diabetic animals treated with insulin. For studies of hormone secretion, labeled thyroid glands were cultured in vitro in medium containing 1 mM mononitrotyrosine. The rate of the hydrolysis of labeled thyroglobulin was measured as the proportion of 131I-labeled iodotyrosines and 131I-labeled iodothyronines recovered at the end of culture and was used as an index of thyroid secretion. TSH in vivo stimulated the rate of thyroglobulin hydrolysis for 6 h, with a peak occurring after 2 h. The diabetic mice had a diminished response to TSH, which improved on treatment with insulin. The addition of TSH and insulin to the culture medium significantly increased the rate of thyroglobulin hydrolysis in glands of diabetic mice over that resulting from the addition of dibutyryl cAMP alone. The generation of thyroidal cAMP in response to TSH was higher in diabetic mice than in controls. The rise in plasma T4 and T3 2 h after the administration of TSH was less in diabetic mice than in control mice or diabetic mice treated with insulin. Our studies, therefore, indicate that the thyroidal response to TSH is decreased in diabetes mellitus. The defect appears to be at a step beyond the generation of cAMP.

  9. Monoclonal antibody-targeted radiotherapy of renal cell carcinoma using a nude mouse model

    SciTech Connect

    Chiou, R.K.; Vessella, R.L.; Limas, C.; Shafer, R.B.; Elson, M.K.; Arfman, E.W.; Lange, P.H.


    Radiation dosimetry and monoclonal antibody (MAB)-targeted radiotherapy studies were performed to evaluate the feasibility of using tumor-preferential MAB as targeting agents for internal radiotherapy of renal cell carcinoma (RCC). Two human RCC xenograft lines, TK-177G and TK-82, were established in nude mice and studied using MAB A6H as a targeting agent. This MAB has previously demonstrated excellent in vivo localization to RCC xenografts. Two doses of A6H (13 to 19 micrograms) labeled with iodine 131 (110 to 130 microCi) caused the tumor to regress or arrested the tumor growth in both xenografts. Similar doses (18 to 43 micrograms; 120 microCi) of /sup 131/I-labeled control MAB AFP-22 or of unlabeled A6H did not inhibit tumor growth. While most mice in the control groups had tumors greater than 250 mg in weight by day 43, none of the tumors in mice treated with /sup 131/I-labeled A6H grew to that size during the 3-month observation period. Sequential computerized scintigraphy was used to calculate the amount of radioisotope localized in tumor versus normal mouse tissue. Therapeutic doses of /sup 131/I-labeled A6H delivered a median calculated radiation dose of 38 cGy/microCi (range, 28 to 57) injected dose to RCC xenografts, and a median of 0.9 cGy/microCi to normal mouse tissues. These findings suggest that A6H is able to target radioisotopes highly specifically to RCC and achieve a therapeutic effect in the experimental setting.

  10. Iodine-131-labeled MAb F(ab')2 fragments are more efficient and less toxic than intact anti-CEA antibodies in radioimmunotherapy of large human colon carcinoma grafted in nude mice

    SciTech Connect

    Buchegger, F.; Pelegrin, A.; Delaloye, B.; Bischof-Delaloye, A.; Mach, J.P. )


    During one week, beginning 18 days after transplantation, nude mice bearing human colon carcinoma ranging from 115 to 943 mm3 (mean 335 mm3) were treated by repeated intravenous injections of either iodine-131-({sup 131}I) labeled intact antibodies or {sup 131}I-labeled corresponding F(ab')2 fragments of a pool of four monoclonal antibodies (MAbs) directed against distinct epitopes of carcinoembryonic antigen (CEA). Complete tumor remission was observed in 8 of 10 mice after therapy with F(ab')2 and 6 of the animals survived 10 mo in good health. In contrast, after treatment with intact MAbs, tumors relapsed in 7 of 8 mice after remission periods of 1 to 3.5 mo despite the fact that body weight loss and depression of peripheral white blood cells, symptoms of radiation toxicity, and the calculated radiation doses for liver, spleen, bone, and blood were increased or equal in these animals as compared to mice treated with F(ab')2.

  11. Radionuclide therapy using nanoparticle of 131I-Lactosome in combination with percutaneous ethanol injection therapy

    NASA Astrophysics Data System (ADS)

    Hara, Eri; Makino, Akira; Kurihara, Kensuke; Ueda, Motoki; Hara, Isao; Kawabe, Takashi; Yamamoto, Fumihiko; Ozeki, Eiichi; Togashi, Kaori; Kimura, Shunsaku


    We present here a radionuclide therapy using nanoparticle of 131I-labeled Lactosome (131I-Lactosome) is effective as a tumor therapy when combined with the local therapy of the percutaneous ethanol injection therapy (PEIT). A mixture of 131I-labeled poly( l-lactic acid)30 and amphiphilic block polymer of poly(sarcosine)64- block-poly( l-lactic acid)30 was dispersed in saline to generate polymeric micelles of 131I-Lactosome (the diameter of 46 nm with PDI of 0.057). Mice were transplanted with murine mammary cancer (4T1) cells, and a relatively small amount of ethanol was injected percutaneously to the tumor region, followed by intravenous administration of 131I-Lactosome (2.0 × 102 MBq/kg). At 16 days after the 131I-Lactosome dosage with PEIT, the relative tumor volume (RTV) was suppressed as low as 5.32 ± 1.06, while the RTV values became significant to be 15.9 ± 5.0 with PEIT alone and 17.4 ± 3.9 with 131I-Lactosome alone. Indeed, 131I-accumulation in the transplanted tumor region at 48 h after 131I-Lactosome dosage became three times higher with PEIT than that without PEIT. The nanoparticle-based radionuclide therapy in combination with PEIT was, therefore, effective in suppression of tumor proliferation.

  12. Testing different storage conditions for (99m)Tc-MAG3 kit: can hot fractioning reduce the cost per unit dose?


    Kiratli, Pinar Ozgen; Salanci, Bilge Volkan


    Since its release for routine clinical use, (99m)Tc-mercaptoacetyltriglycine (MAG3) has become an important alternative to (131)I-labeled orthoiodohippuran. The cold kit for MAG3 is expensive, especially in developing countries. Therefore, unique storage conditions should be provided for cost reduction. Cold fractioning is a well-known procedure but has special requirements, such as a nitrogen tank and a laminar flow hood. The aim of this study was to prolong the shelf life of (99m)Tc-labeled MAG3 by a hot fractioning method, which separates the patient doses after (99m)Tc labeling. The radiochemical purity of the (99m)Tc-labeled MAG3 kit was tested under different storage conditions. Hot fractioning of the (99m)Tc-labeled MAG3 kit was found to be a possible alternative to cold fractioning for routine clinical studies.

  13. Tissue distribution and radiation dosimetry of astatine-211-labeled chimeric 81C6, an alpha-particle-emitting immunoconjugate.


    Zalutsky, M R; Stabin, M G; Larsen, R H; Bigner, D D


    A paired-label study was performed in athymic mice bearing subcutaneous D-54 MG human glioma xenografts to compare the localization of human/mouse anti-tenascin chimeric antibody 81C6 labeled by reaction with N-succinimidyl 3-[211At]astatobenzoate and N-succinimidyl 3-[131I]iodobenzoate. Over the 48-h observation period, the distribution of 211At- and 131I-labeled antibody were quite similar in tumor and normal tissues except stomach. These data were used to calculate human radiation doses for both intravenously and intrathecal administered 211At-labeled chimeric 81C6 using a quality factor of 5 for alpha-emissions.

  14. Impromidine-induced changes in the permeability of the blood-brain barrier of normotensive and spontaneously hypertensive rats

    SciTech Connect

    Boertje, S.B.; Le Beau, D.; Ward, S. )


    Previous studies suggested histamine receptors mediate changes in the cerebrovascular permeability of rats. To test this, we investigated the effects of impromidine, a specific agonist at the histamine H2-receptor, on blood pressure and permeability of the blood-brain barrier (BBB). Impromidine produced dose-dependent hypotension in Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Two higher doses of impromidine increased BBB permeability to 99mTc-sodium pertechnetate in WKY rats; however, two lower doses decreased permeability in SHR rats. All doses of impromidine increased cerebrovascular permeability to 131I-labeled serum albumin in both species. Doses of the drug were 100 times greater than those required to produce similar alterations using histamine.

  15. Sandmeyer reaction repurposed for the site-selective, non-oxidizing radioiodination of fully-deprotected peptides: studies on the endogenous opioid peptide α-neoendorphin.


    Pickett, Julie E; Nagakura, Kunihiko; Pasternak, Anna R; Grinnell, Steven G; Majumdar, Susruta; Lewis, Jason S; Pasternak, Gavril W


    Standard radioiodination methods lack site-selectivity and either mask charges (Bolton-Hunter) or involve oxidative reaction conditions (chloramine-T). Opioid peptides are very sensitive to certain structural modifications, making these labeling methods untenable. In our model opioid peptide, α-neoendorphin, we replaced a tyrosyl hydroxyl with an iodine, and in cell lines stably expressing mu, delta, or kappa opioid receptors, we saw no negative effects on binding. We then optimized a repurposed Sandmeyer reaction using copper(I) catalysts with non-redoxing/non-nucleophilic ligands, bringing the radiochemical yield up to around 30%, and site-selectively incorporated radioactive iodine into this position under non-oxidizing reaction conditions, which should be broadly compatible with most peptides. The (125)I- and (131)I-labeled versions of the compound bound with high affinity to opioid receptors in mouse brain homogenates, thus demonstrating the general utility of the labeling strategy and of the peptide for exploring opioid binding sites.

  16. Characterisation and radioimmunotherapy of L19-SIP, an anti-angiogenic antibody against the extra domain B of fibronectin, in colorectal tumour models.


    El-Emir, E; Dearling, J L J; Huhalov, A; Robson, M P; Boxer, G; Neri, D; van Dongen, G A M S; Trachsel, E; Begent, R H J; Pedley, R B


    Angiogenesis is a characteristic feature of tumours and other disorders. The human monoclonal antibody L19- SIP targets the extra domain B of fibronectin, a marker of angiogenesis expressed in a range of tumours. The aim of this study was to investigate whole body distribution, tumour localisation and the potential of radioimmunotherapy with the L19-small immunoprotein (SIP) in colorectal tumours. Two colorectal tumour models with highly different morphologies, the SW1222 and LS174T xenografts, were used in this study. Localisation and retention of the L19-SIP antibody at tumour vessels was demonstrated using immunohistochemistry and Cy3-labelled L19-SIP. Whole body biodistribution studies in both tumour models were carried out with (125)I-labelled L19-SIP. Finally, (131)I-labelled antibody was used to investigate the potential of radioimmunotherapy in SW1222 tumours. Using immunohistochemistry, we confirmed extra domain B expression in the tumour vasculature. Immunofluorescence demonstrated localisation and retention of injected Cy3-labelled L19-SIP at the abluminal side of tumour vessels. Biodistribution studies using a (125)I-labelled antibody showed selective tumour uptake in both models. Higher recorded values for localisation were found in the SW1222 tumours than in the LS174T (7.9 vs 6.6 %ID g(-1)), with comparable blood clearance for both models. Based on these results, a radioimmunotherapy study was performed in the SW1222 xenograft using (131)I-Labelled L19-SIP (55.5 MBq), which showed selective tumour uptake, tumour growth inhibition and improved survival. Radio- and fluorescence-labelled L19-SIP showed selective localisation and retention at vessels of two colorectal xenografts. Furthermore, (131)I-L19-SIP shows potential as a novel treatment of colorectal tumours, and provides the foundation to investigate combined therapies in the same tumour models.

  17. Specific PET Imaging Probes for Early Detection of Prostate Cancer Metastases

    DTIC Science & Technology


    MALDI - TOF mass spectrometry (see Supplementary information). Benzyl-2- acetamido-2-deoxy-a-D-galactopyranoside (BG), Swainsonine (SW) and HPLC and MALDI - TOF mass spectrometry (see Supplementary information). Benzyl-2-acetamido-2-deoxy-a-D-galactopyranoside (BG), swainsonine (SW) and...that the involvement of Rho protein in the uptake of R11 peptide by prostate cells may be minimal. GAGs and anionic polymers have been shown to affect

  18. Differences in the hydrolysis of lactose and other substrates by beta-D-galactosidase from Kluyveromyces lactis.


    Kim, S H; Lim, K P; Kim, H S


    The hydrolysis of o-nitrophenyl galactopyranoside and lactose by beta-D-galactosidase from Kluyveromyces lactis was enhanced by the addition of Mg2+ and Mn2+, but the rates of activation by each metal on both substrates were not the same. The Co2+, Zn2+, and Ni2+ activated the o-nitrophenyl galactopyranoside-hydrolyzing activity of the enzyme, but these same metals inhibited the lactose-hydrolyzing activity. The addition of Mg2+ and EDTA to the assay buffer increased the hydrolysis of o-nitrophenyl galactopyranoside and lactose at different rates. The responses of o-nitrophenyl galactopyranoside and lactose to the enzyme activity were different as a function of pH. The hydrolyzing activity toward both substrates also was influenced by the concentration of the phosphate in the assay buffer. However, the profile of the enzyme activity toward each substrate was different as a function of concentration. Because the assay of beta-galactosidase using o-nitrophenyl galactopyranoside is fast and convenient, the estimation of lactose-hydrolyzing activity of the enzyme has frequently been made based on the assay of o-nitrophenyl galactopyranoside hydrolysis. As shown in this study, a slight change in the conditions of the assay system and the enzyme application may cause changes in the ability of the enzyme to hydrolyze both lactose and o-nitrophenyl galactopyranoside. The change in o-nitrophenyl galactopyranoside-hydrolyzing activity is not always consistent with that of the lactose-hydrolyzing activity under the given condition, which may cause an inaccurate estimation of the enzyme activity in the enzyme preparation as well as in actual applications of the enzyme.

  19. Isolation and structural characterization of unusual pyranoanthocyanins and related anthocyanins from Staghorn sumac (Rhus typhina L.) via UPLC-ESI-MS, (1)H, (13)C, and 2D NMR spectroscopy.


    Kirby, Christopher W; Wu, Tao; Tsao, Rong; McCallum, Jason L


    The six major anthocyanins found in the burgundy coloured fruits of Staghorn sumac (Rhus typhina L.) were isolated and the structures of four compounds were determined by NMR spectroscopic methods as being: 7-O-methyl-delphinidin-3-O-(2″galloyl)-β-d-galactopyranoside; 7-O-methyl-cyanidin-3-O-(2″galloyl)-β-d-galactopyranoside; 7-O-methyl-delphinidin-3-O-(2″'galloyl)-β-d-galactopyranoside-4-vinyl-catechol-3″-O-β-d-glucopyranoside; and 7-O-methyl-cyanidin-3-O-(2″'galloyl)-β-d-galactopyranoside-4-vinyl-catechol-3″-O-β-d-glucopyranoside, respectively. Additionally, two related anthocyanin compounds, cyanidin-3-O-(2″galloyl)-β-d-galactopyranoside and 7-O-methyl-cyanidin-3-O-β-d-galactopyranoside were also recovered, with NMR spectroscopic values closely matching previous reports from other plant species. The prevalence of 7-O-methyl anthocyanins and their galloylated derivatives in sumac is highly unusual, and warrants special attention. Additionally, the in planta occurrence of two 7-O-methyl-pyranoanothocyanin-vinyl-catechol aglycones, Sumadin A and Sumadin B, and their derivatives is noted. To our knowledge, E-ring glycosylated vinyl-catechol pyranoanthocyanins were previously unknown.

  20. Inhibitory effect on TNF-α-induced IL-8 secretion in HT-29 cell line by glyceroglycolipids from the leaves of Ficus microcarpa.


    Kiem, Phan Van; Minh, Chau Van; Nhiem, Nguyen Xuan; Cuong, Nguyen Xuan; Tai, Bui Huu; Quang, Tran Hong; Anh, Hoang Le Tuan; Yen, Pham Hai; Ban, Ninh Khac; Kim, Seung Hyun; Xin, Mingjie; Cha, Ji-Yun; Lee, Young-Mi; Kim, Young Ho


    Bioassay-guided fractionation based on the anti-inflammatory activity of a methanol extract of Ficus microcarpa leaves led to the isolation of seven galactolipids: 2(S)-3-O-octadeca-9Z,12Z,15Z-trienoylglyceryl-O-β-D-galactopyranoside (1), (2S)-2,3-O-dioctadeca-9Z,12Z,15Z-trienoylglyceryl-O-β-D-galactopyranoside (2), (2S)-2,3-O-dioctadeca-9Z,12Z-dienoylglyceryl-O-β-D-galactopyranoside (3), (2S)-3-O-octadeca-9Z,12Z,15Z-trienoylglyceryl-6'-O-(α-D-galactopyranosyl)-β-D-galactopyranoside (4), (2S)-2,3-O-dioctadeca-9Z,12Z,15Z-trienoylglyceryl-6'-O-(α-D-galactopyranosyl)-β-D-galactopyranoside (5), gingerglycolipid B (6), and (2S)-2,3-O-dioctadeca-9Z,12Z-dienoylglyceryl-6'-O-(α-D-galactopyranosyl)-β-D-galactopyranoside (7). Their chemical structures were elucidated by mass, 1D-, and 2D-NMR spectroscopic methods as well as chemical methods. The antiinflammatory effect of these compounds on TNF-α induced IL-8 secretion in the HT-29 cell line was evaluated. All above galactolipids showed significant inhibition ranging 40% at a concentration of 50 μM. The results suggest that galactolipids from the leaves of F. microcarpa may be used as potent anti-inflammatory agents.

  1. Iodophenylpentadecanoic acid-myocardial blood flow relationship during maximal exercise with coronary occlusion

    SciTech Connect

    Caldwell, J.H.; Martin, G.V.; Link, J.M.; Krohn, K.A.; Bassingthwaighte, J.B. )


    Imaging {sup 123}I-labeled iodophenylpentadecanoic acid (IPPA) uptake and clearance from the myocardium following exercise has been advocated as a means of detecting myocardial ischemia because fatty acid deposition is enhanced and clearance prolonged in regions of low flow. However, normal regional myocardial blood flows are markedly heterogeneous, and it is not known how this heterogeneity affects regional metabolism or substrate uptake and thus image interpretation. In five instrumented dogs running at near maximal workload on a treadmill, {sup 131}I-labeled IPPA and 15-micron 46Sc microspheres were injected into the left atrium after 30 sec of circumflex coronary artery occlusion. Microsphere and IPPA activity were determined in 250 mapped pieces of myocardium of approximately 400 mg. Myocardial blood flows (from microspheres) ranged from 0.05 to 7.6 ml/min/g. Deposition of IPPA was proportional to regional flows (r = 0.83) with an average retention of 25%. The mean endocardial-epicardial ratio for IPPA (0.90 {plus minus} 0.43) was similar to that for microspheres (0.94 {plus minus} 0.47; p = 0.08). Thus, initial IPPA deposition during treadmill exercise increases in proportion to regional myocardial blood flow over a range of flows from very low to five times normal.

  2. Measurement and modeling of iodine volatility above irradiated CsI solutions

    SciTech Connect

    Evans, G.J.


    Any release of radioiodine to the environment following a reactor accident depends to a large extent on its volatility within a containment structure. A common measure of iodine volatility is the water-air volumetric iodine partition coefficient (IPC), defined as the ratio of aqueous to airborne radioiodine concentrations. The impact of pH and total iodine concentration on volatility is evaluated through experiments and modeling to establish the relevant trends and improve the understanding of the underlying mechanisms. The model consists of kinetic expressions for 125 reactions. The IPC is evaluated experimentally by irradiating, at 0.25 kGy/h and 25 C, {sup 131}I-labeled CsI solutions ranging in concentration from 10{sup {minus}8} to 10{sup {minus}4} M and in pH from 3 to 12. Both the experiments and the modeling indicate that under acidic conditions, the IPC for 10{sup {minus}6} M solutions is substantially higher than that for 10{sup {minus}5} M solutions. The predicted dependence of the IPC on pH for acidic 10{sup {minus}5} M solutions is in good agreement with that observed experimentally. However, substantial divergence occurred for more dilute solutions and for basic pH conditions. It is speculated that under these conditions, atomic iodine may contribute substantially to the overall volatility; adding atomic iodine volatility to the model is found to greatly improve the agreement.

  3. Methods of assessment of thrombosis in vivo

    SciTech Connect

    Dewanjee, M.K.


    The contributions of platelets and clotting factors in thrombosis on injured vessel and cardiovascular prostheses have been quantified with several tracers. Thrombus formation in vivo could be measured semiquantitatively in animal models and humans with /sup 111/In-labeled platelets, /sup 123/I- and /sup 131/I-labeled fibrinogen, /sup 111/In-labeled antibody to the fibrinogen receptor on the platelet membrane and to fibrin. Thrombus localization by imaging was possible for large thrombus in vessel with deep injury of thrombogenic surface in the acute phase. A single layer of adherent platelets could not be imaged, due to the high background radioactivity present in blood. Thrombogenicity of grafts was compared with that of contralateral vessel. The dynamic process of platelet deposition could be followed accurately using the in vivo imaging technique. In addition, in vitro quantification permits determination of platelet and fibrin density and of the number of fibrin monomers per platelet in thrombus. The roles of prostacyclin, thromboxane inhibitors, and nonsteroidal antiinflammatory drugs have also been evaluated in animals models and humans. The tracer techniques thus provide invaluable information about platelet-fibrin deposition, its organization and dissolution, and for development of less thrombogenic surfaces for use in cardiovascular prostheses. 53 references.

  4. Uptake and depuration of 131I by the edible periwinkle Littorina littorea: uptake from seawater.


    Vives i Batlle, J; Wilson, R C; McDonald, P; Parker, T G


    Uptake and depuration experiments for the edible periwinkle Littorina littorea have been performed using 131I-labelled seawater. Throughout the experimental phase the winkles were fed on unlabelled Chondrus crispus. 131I concentrations in winkles during uptake followed linear first-order kinetics with an uptake half-time of 11 days, whereas for depuration a triphasic sequence with biological half-lives of 4, 23 and 56 days was determined. In general, iodine turnover in winkles via labelled seawater appears to be slower than observed for other molluscs (2-3 days). Most of the activity prior to and after depuration is found to be in the shell, with indications that shell and soft parts accumulate and depurate 131I at a similar rate. The operculum displays the highest specific activity of all fractions with a concentration factor of 750 l kg(-1). Concentration factors for whole winkle, shell, soft parts and digestive gland are in the order of 40-60 l kg(-1), higher than the IAEA recommended CF value for iodine in molluscs of 10 l kg(-1). The 131I CF in winkles is closer to that of the conservative radionuclides 99Tc and 137Cs than the CF of the particle reactive radionuclides (239,240)Pu and 241Am.

  5. Hepatic injury after whole-liver irradiation in the rat

    SciTech Connect

    Geraci, J.P.; Jackson, K.L.; Mariano, M.S.; Leitch, J.M.


    Radiation-induced hepatic injury in rats, which is characterized by marked ascites accompanied by liver necrosis, fibrosis, and vein lesions, is described in this study. These adverse sequelae are produced within 30 days after irradiation if there is surgical removal of two-thirds of the liver immediately after whole-liver irradiation. The LD/sub 50/30/ day and median survival time after liver irradiation and two-thirds partial hepatectomy is 24 Gy and 17 days, respectively. Death is preceded by reduction in liver function as measured by (/sup 131/I)-labeled rose bengal clearance. Prior to death, liver sepsis and endotoxemia were detected in most irradiated, partially hepatectomized animals. Pretreatment of the animals with endotoxin and/or antibiotic decontamination of the GI tract resulted in increased survival time, but no irradiated, partially hepatectomized animal survived beyond 63 days. This suggests that sepsis and endotoxemia resulting from the bacteria in the intestine are the immediate cause of death after 30-Gy liver irradiation and partial hepatectomy. It is concluded that the hepatectomized rat model is an economical and scientifically manageable experimental system to study a form of radiation hepatitis that occurs in compromised human livers.

  6. Astatine-211 labeling of internalizing anti-EGFRvIII monoclonal antibody using N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate.


    Reist, C J; Foulon, C F; Alston, K; Bigner, D D; Zalutsky, M R


    Monoclonal antibodies (MAbs) such as the anti-epidermal growth factor variant III (EGFRvIII) MAb L8A4 are rapidly internalized, which can lead to rapid loss of radioactivity from the tumor cell. The aim of this study was to evaluate the potential utility of N-succinimidyl 5-[211At]astato-3-pyridinecarboxylate ([211At]SAPC) for labeling murine L8A4 with 211At. SAPC was synthesized by astatodestannylation of N-succinimidyl 5-tri-n-butylstannyl 3-pyridinecarboxylate and then coupled to L8A4 in approximately 50% yield. The affinity and immunoreactive fraction for 211At-labeled L8A4 were comparable to those obtained when the MAb was labeled with 131I via N-succinimidyl 5-[131I]iodo-3-pyridinecarboxylate (SIPC). Paired-label comparisons of the 211At- and 131I-labeled MAbs demonstrated similar internalization and catabolism by EGFRvIII-positive cells in vitro, and with the exception of the stomach, similar tissue distribution in athymic mice with EGFRvIII-expressing U87MGdeltaEGFR xenografts. These results suggest that SAPC may be a useful reagent for labeling L8A4, and possibly other internalizing proteins, with 211At.

  7. A physiological systems model for iodine for use in radiation protection

    SciTech Connect

    Leggett, Richard Wayne


    This paper summarizes the biokinetic database for iodine in the human body and proposes a biokinetic model for use in dose assessments for radioiodine. The model unifies and extends existing physiological systems models describing three subsystems of the iodine cycle in the body: circulating (extrathyroidal) inorganic iodide; thyroidal iodine (trapping and organic binding of iodide, and synthesis, storage, and secretion of thyroid hormones); and extrathyroidal organic iodine. Thyroidal uptake of iodide is described as a function of daily stable iodine intake and requirements for thyroid hormones. Baseline parameter values are developed for adults with typical iodine intakes and hormone requirements. Estimated thyroid doses derived from the baseline parameter values and reference thyroid weights are higher than values predicted by the current model of the International Commission on Radiological Protection (ICRP) for adults for intake of iodine isotopes with half-lives up to a few hours but consistent with ICRP predictions for longer-lived isotopes. For nearly all iodine isotopes, the proposed model yields order-of-magnitude differences from the ICRP s current iodine model for adults for stomach wall, salivary gland, and kidneys. Dose estimates for intravenously injected 131I-labeled thyroid hormones based on the present model differ substantially from current ICRP values for adult patients for some organs, including the thyroid. Subsequent studies will address age-specific biokinetics of iodine, reduction of doses from radioiodine due to thyroid blocking, and effects of dietary iodine levels and thyroid hormone requirements on thyroid doses from radioiodine.

  8. Measurement of lung fluid volumes and albumin exclusion in sheep

    SciTech Connect

    Pou, N.A.; Roselli, R.J.; Parker, R.E.; Clanton, J.A.; Harris, T.R. )


    A radioactive tracer technique was used to determine interstitial diethylenetriaminepentaacetic acid (DTPA) and albumin distribution volume in sheep lungs. {sup 125}I- and/or {sup 131}I-labeled albumin were injected intravenously and allowed to equilibrate for 24 h. {sup 99m}Tc-labeled DTPA and {sup 51}Cr-labeled erythrocytes were injected and allowed to equilibrate (2 h and 15 min, respectively) before a lethal dose of thiamylal sodium. Two biopsies (1-3 g) were taken from each lung and the remaining tissue was homogenized for wet-to-dry lung weight and volume calculations. Estimates of distribution volumes from whole lung homogenized samples were statistically smaller than biopsy samples for extravascular water, interstitial {sup 99m}Tc-DTPA, and interstitial albumin. The mean fraction of the interstitium (Fe), which excludes albumin, was 0.68 +/- 0.04 for whole lung samples compared with 0.62 +/- 0.03 for biopsy samples. Hematocrit may explain the consistent difference. To make the Fe for biopsy samples match that for homogenized samples, a mean hematocrit, which was 82% of large vessel hematocrit, was required. Excluded volume fraction for exogenous sheep albumin was compared with that of exogenous human albumin in two sheep, and no difference was found at 24 h.

  9. Relative rates of albumin equilibration in the skin interstitium and lymph during increased permeability

    SciTech Connect

    Powers, M.R.; Wallace, J.R.; Bell, D.R.


    The initial equilibration of /sup 125/I-labelled albumin between the vascular and extravascular compartments was studied in hindpaw heel skin of anesthetized rabbits. Bradykinin (0.3 was infused into a small branch of the femoral artery. A second group of rabbits served as control. Following bradykinin, prenodal popliteal lymph flow was 4 times control flow. The lymph-to-plasma concentration ratios for total protein and albumin were, respectively, 60% and 50% larger than control. Tissue albumin concentration was twice control. After reaching a steady, elevated lymph flow, tracer albumin was infused to maintain plasma activity constant for 3 hrs. The plasma volume in tissue samples was measured using /sup 131/I-labeled albumin injected 10 min before ending the experiment. Endogenous albumin was measured in plasma, lymph, and tissue samples using rocket electroimmunoassay. After 3 hrs of tracer infusion, lymph specific activity was 3 times greater than control. In the control group, plasma albumin equilibrated more rapidly with lymph than with tissue (p < 0.05). Following bradykinin, extravascular specific activity was 4 times control, resulting in lymph and tissue equilibrating with plasma at similar rates. Thus, increasing capillary permeability causes the extravascular albumin mass to behave as if distributed in a single compartment.

  10. Radioimmunoimaging of pneumocystis carinii infection in rats

    SciTech Connect

    Vallabhajosula, S.; Shane, L.B.; Goldsmith, S.J.; Lipszyc, H.; Walzer, P.


    Pneumocystis carinil pneumonia (PCP) is seen in patients with impaired immunity due to chemotherapeutic suppression or to a primary disorder, congenital or AIDS. Although radiogallium imaging has been helpful in the workup of PCP, it is non-specific. Since there is no early specific non-invasive method to diagnose PCP, the authors are developing an imaging technique using radiolabeled antibodies. Fulminant PCP was induced in rats by injecting cortisone, 20mg 2-3 times/wk for 8 wks. PC cells isolated from rat lung were injected into rabbits. The antiserum thus derived was separated and purified using Protein-A bound sepharose column with identification of IgG by polyacrylamide gel electrophoresis. Both rabbit antipneumocystis antibodies and purified IgG(Sigma) were iodinated with I-131 to a high specific activity ( using a lactoperoxidase method. /sup 131/I-labeled specific and non-specific IgG were injected into rats with PC infection and imaged with an Anger camera. After sacrifice, I-131 activity/gram tissue (lung, liver, heart) was determined and expressed as organ ratios. An increased uptake of specific antibody in lungs of rats with PCP was demonstrated by organ counting and imaging. This increase was not seen in normal controls or rats injected with non-specific IgG. These data provide a basis for radioimmunoimaging of infectious diseases.

  11. The impact of tumor size on the efficacy of monoclonal antibody-targeted radiotherapy: studies using a nude mouse model with human renal cell carcinoma xenografts

    SciTech Connect

    Chiou, R.K. )


    Monoclonal antibody (Mab)-targeted radiotherapy is a unique approach in cancer therapy. Multiple factors affect the success of treatment. Internal radiation dosimetry and mini-dose Mab-targeted radiotherapy studies reveal that tumor size affects the efficacy of treatment. For tumors with calculated weight greater than 400 mg., intravenous administration of 131I-labeled A6H or A6H-C5H combination delivers significantly less tumor radiation dose (2070 +/- 580 cGy/100 microCi) than those for tumors of weight less than 200 mg. (5260 +/- 2460 cGy/100 microCi). In the mini-dose (an average of 73 to 86 microCi) Mab-targeted radiotherapy study of 109 mice with small tumors (six, 12, or 19 days after implantation), tumors 12 days after implantation (approximately 60 mg. in weight) showed regression in all mice and in 62% of mice gross tumor elimination was observed. In contrast, mini-dose therapy at day 19 (tumor weights approximately 170 mg.) resulted in tumor regression and tumor elimination rates of 33% and 17% respectively. These studies suggest that Mab-targeted radiotherapy is more suitable for treating small tumors.

  12. Uptake and distribution of specific and control monoclonal antibodies in subcutaneous xenografts following intratumor injection

    SciTech Connect

    Rowlinson-Busza, G.; Bamias, A.; Krausz, T.; Epenetos, A.A. )


    Nude mice bearing s.c. xenografts of the human colon adenocarcinoma HT29 were given intratumor injections of a mixture of 125I-labeled specific antibody (AUA1) and 131I-labeled control antibody (HMFG1), or with the labels reversed. After dissection at 1 and 4 h postadministration, both specific and control antibodies had 47-63% of the injected dose (% ID) in the tumor. By 24 h, the tumor contained 43 {plus minus} 11% ID of AUA1 which persisted at around this level for 5 days and remained at nearly 20% ID at 18 days. In contrast, the HMFG1 activity was 23 {plus minus} 9% ID at 24 h, which continued to fall and was less than 5% ID by 7 days. Normal organ levels were less than 2% ID/g for both antibodies, with HMFG1 being higher than AUA1 at all times, resulting in specificity indices greater than 20 by 5 days. Autoradiography of tumors removed 2 h postinjection of 125I-labeled AUA1 or HMFG1 showed high levels of antibody at the injection site. At 48 h and 7 days postinjection, the specific antibody was bound to the surface of tumor cells in islands remote from the injection site, whereas the control antibody was found only in the stroma and blood vessels, or as diffuse nonspecific uptake. These data indicate that intratumor injection of radiolabeled monoclonal antibodies may achieve high radiation doses in accessible tumors without systemic irradiation.

  13. The presence of MOMA-2+ macrophages in the outer B cell zone and protection of the splenic micro-architecture from LPS-induced destruction depend on secreted IgM.


    Fischer, Michael B; Rüger, Beate; Vaculik, Christine; Becherer, Alexander; Wadsak, Wolfgang; Yanagida, Genya; Losert, Udo M; Chen, Jianzhu; Carroll, Michael C; Eibl, Martha M


    The role secretory IgM has in protecting splenic tissue from LPS-induced damage was assessed in mice incapable of secreting IgM but able to express surface IgM and IgD. Within seconds after LPS challenge, 99% of the (131)I-labeled LPS was found in the liver and the spleen of both sIgM-deficient and wild-type mice. In the spleen FITC-labeled LPS was found on the surface of 2F8(+) scavenger receptor macrophages localized in the outer marginal zone, while none of the labeled LPS could be detected on marginal zone ER-TR9(+) and MOMA-1(+) macrophages. An additional population of macrophages, MOMA-2(+), were capable of producing C3 locally in the T and B cell zone after LPS challenge. Local C3 production was regulated, as no C3 was found in splenic tissue of unchallenged mice. Interestingly, in the absence of circulating and locally produced secretory IgM, MOMA-2(+) macrophages of the T and B cell zone failed to establish an additional ring of C3-producing macrophages in the outer B cell zone close to the marginal zone upon LPS challenge. The consequence was a massive destruction of the microarchitecture of the spleen where marginal zones disorganized, lymphoid follicles and T cell zones disrupted and follicular DC (FDC) networks disappeared.

  14. Tumor immunoscintigraphy by means of radiolabeled monoclonal antibodies: Multicenter studies of the Italian National Research Council--Special Project Biomedical Engineering

    SciTech Connect

    Siccardi, A.G. )


    Four radioimmunopharmaceuticals ({sup 99m}Tc- and 111In-labeled anti-melanoma and {sup 111}In- and {sup 131}I-labeled anti-carcinoembryonic antigen F(ab')2 fragments derived from monoclonal antibodies 225.28S and F023C5) were developed by means of a collaborative effort coordinated by the Italian National Research Council, Special Project Biomedical Engineering. After appropriate pilot studies, the radioimmunopharmaceuticals, prepared by Sorin Biomedica (Saluggia, Italy), were distributed to 31 Nuclear Medicine departments in Italy and in 10 other European countries within the framework of three immunoscintigraphy multicenter studies. A total of 1245 patients were studied, 898 of whom carried 1725 documented tumor lesions; 1596 of 2193 tumor lesions (468 of which were previously unknown) were imaged by immunoscintigraphy in 785 of 990 lesion-bearing patients. Among the occult lesions, 173 were imaged in 92 patients admitted to the study as lesion-free patients. The results have been analyzed in terms of the reliability, reproducibility, and diagnostic usefulness of the method and of each immunoradiopharmaceutical.

  15. Experimental radioimmunotherapy of a xenografted human colonic tumor (GW-39) producing carcinoembryonic antigen

    SciTech Connect

    Goldenberg, D.M.; Gaffar, S.A.; Bennett, S.J.; Beach, J.L.


    Experiments were undertaken to evaluate the antitumor effects of 131I-labeled goat antibody immunoglobulin G prepared against carcinoembryonic antigen in hamsters bearing the carcinoembryonic antigen-producing GW-39 human colonic carcinoma. At a single injection of 1 mCi 131I and higher, a marked growth inhibition of GW-39 tumors, as well as a considerable increase in the survival time of the tumor-bearing hamsters, could be achieved. At a dose of 1 mCi, the radioactive affinity-purified antibody appeared to be superior to radioactive normal goat immunoglobulin G in influencing tumor growth and survival time, but no significant difference could be seen at the higher dose of 2 mCi given. Radiobiological calculations indicated that the tumors received, at up to 20 days after therapy, 1325 rads for the specific antibody and only 411 rads for the normal immunoglobulin G preparation. These findings encourage the further evaluation of antibodies to tumor markers for isotopic cancer therapy.

  16. Antibody guided diagnosis and therapy of brain gliomas using radiolabeled monoclonal antibodies against epidermal growth factor receptor and placental alkaline phosphatase

    SciTech Connect

    Kalofonos, H.P.; Pawlikowska, T.R.; Hemingway, A.; Courtenay-Luck, N.; Dhokia, B.; Snook, D.; Sivolapenko, G.B.; Hooker, G.R.; McKenzie, C.G.; Lavender, P.J. )


    Twenty-seven patients with brain glioma were scanned using {sup 123}I-labeled monoclonal antibodies against epidermal growth factor receptor (EGFR1) or placental alkaline phosphatase (H17E2). Successful localization was achieved in 18 out of 27 patients. Eleven out of 27 patients were also studied using a nonspecific control antibody (11.4.1) of the same immunoglobulin subclass and observable tumor localization was also achieved in five patients. The specificity of targeting was assessed by comparing images obtained with specific and nonspecific antibodies and by examining tumor and normal tissue biopsies after dual antibody administration. Ten patients with recurrent grade III or IV glioma who showed good localization of radiolabeled antibody were treated with 40-140 mCi of {sup 131}I-labeled antibody delivered to the tumor area intravenously (n = 5) or by infusion into the internal carotid artery (n = 5). Six patients showed clinical improvement lasting from 6 mo to 3 yr. One patient continues in remission (3 yr after therapy), but the other five who responded initially relapsed 6-9 mo after therapy and died. No major toxicity was attributable to antibody-guided irradiation. Targeted irradiation by monoclonal antibody may be clinically useful and should be explored further in the treatment of brain gliomas resistant to conventional forms of treatment.

  17. Intravenous administration of alpha-1-proteinase inhibitor in patients of PiZ and PiM phenotype. Preliminary report

    SciTech Connect

    Moser, K.M.; Smith, R.M.; Spragg, R.G.; Tisi, G.M.


    Nine patients with moderate pulmonary emphysema, six of PiZ phenotype and three of PiM phenotype, have received a single intravenous infusion of alpha-1-proteinase inhibitor (human) (A1PI), in a dose of 60 mg/kg over a 30-minute period. They also received a tracer dose (300 microCi) of /sup 131/I-labeled A1PI. No active or passive immunization against hepatitis was given. No acute toxicity was observed. Compared with baseline data, significant elevations of serum A1PI (measured both antigenically and as anti-elastase activity) occurred, with a serum half-life approximating 110 hours. Bronchoalveolar lavage fluid, obtained 48 hours after infusion, reflected a significant increase in A1PI concentration versus baseline bronchoalveolar lavage fluid values. Serial gamma camera images of the lungs confirmed persistence of enhanced lung radioactivity for several days. Urinary desmosine excretion did not change following A1PI infusion. During the period of follow-up thus far, no patient has had chronic toxicity, results of liver function tests have been stable, and there has been no development of hepatitis B antigen or antibodies to hepatitis B surface or core antigens.

  18. Visualization of metastases from colon carcinoma using an iodine 131-radiolabeled monoclonal antibody

    SciTech Connect

    Leyden, M.J.; Thompson, C.H.; Lichtenstein, M.; Andrews, J.T.; Sullivan, J.R.; Zalcberg, J.R.; McKenzie, I.F.


    A murine monoclonal antibody that reacts with human colonic cancer (250-30.6) was labeled with radioactive iodine (131I) and the antibody was injected intravenously into 15 patients with known metastases originating from carcinoma of the colon (10 cases), malignant melanoma (1), breast (1), pancreas (1), hepatocellular carcinoma (1), and adenocarcinoma of unknown origin (1). Of the patients with metastatic colon carcinoma, there were 19 known deposits as judged by the techniques of clinical examination, x-rays, and scans obtained using sulpha-colloid. Of these 19 deposits, 17 (90%) were found using the 131I-labeled monoclonal antibody. In one case, the primary tumor, previously undiagnosed, was found. In only 1 of the 10 patients was tumor not found and this was due to the subsequent finding that the undifferentiated tumor did not react with antibody. Of the five patients who did not have carcinoma of the colon, three had negative scans, but two were positive. Thus, the technique of immunoscintography can readily detect both primary and metastatic tumors.

  19. Characterization of high titer antithyroglobulin antibodies.


    Costin, A; Klandorf, H; Persselin, J; Van Herle, A J


    Few autoantibodies directed against thyroglobulin (TgAbs) have been fully characterized in man. The present study was designed to characterize TgAbs from patients with unusually high titers (greater than 1:5(12] using tanned red cell hemagglutination technique (TRC). IgG was isolated from the sera of subjects with Hashimoto's thyroiditis (n = 4), subacute thyroiditis (n = 1) and Graves' disease (n = 1) using DEAE-Sephacel chromatography. Isolated TgAbs were substituted as first antibody in a double antibody thyroglobulin (Tg) RIA and the Ka's were determined by Scatchard analysis. Molecular ratios of antibody to antigen, TgAb: Tg, were calculated from quantitative precipitin curves. The clonality of each antibody was determined using agarose isoelectric focusing and 131I labeled Tg as an autoradiographic probe. All six TgAbs were polyclonal. The Ka's were on the order of 10(9)-10(10). In two sera TgAb:Tg ratios of 20:1 and 8:1 were obtained. These results are significant when compared to previously characterized Tg autoantibodies which have been of low titer, low Ka (10(5], and have been directed towards a restricted portion of the Tg molecule (TgAb: Tg ratios of 2:1 to 6:1). In view of their high affinity constants and recognition of a less restricted portion of the Tg molecule, some of the TgAb's with unusually high titers behave more like Tg heteroantibodies than autoantibodies.

  20. Pharmacokinetic studies of mouse monoclonal antibodies to a rat colon carcinoma: I. Comparison of biodistribution in normal rats, syngeneic tumor-bearing rats, or tumor-bearing nude mice

    SciTech Connect

    Laborda, J.; Douillard, J.Y.; Burg, C.; Lizzio, E.F.; Ridge, J.; Levenbook, I.; Hoffman, T. )


    The pharmacokinetics of two iodine-131-({sup 131}I) labeled murine anti-rat colon carcinoma monoclonal antibodies (D3 and E4) were compared in normal Sprague Dawley rats, syngeneic BDIX rats, or nude mice bearing that tumor. Results of antibody uptake after i.v. administration were analyzed in terms of accumulation and localization indices for normal tissues and tumor. Statistically significant differences between rat and mouse tissue biodistribution were found. D3, which reacts in vitro with the tumor and several normal rat tissues, cleared quickly from the blood of rats and was specifically targeted to several normal tissues, notably the lung. Virtually no targeting to the tumor was observed. Nude mice, however, showed a slower blood clearance and specific antibody targeting only in the tumor. Similar results were seen after injection of another antibody, E4, which is tumor-specific in vitro. Data suggest that studies on the xenogeneic nude mouse model may not necessarily be relevant to the choice of monoclonal antibodies for clinical diagnostic imaging or therapy.

  1. Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition

    SciTech Connect

    Chaptal, Vincent; Kwon, Seunghyug; Sawaya, Michael R.; Guan, Lan; Kaback, H. Ronald; Abramson, Jeff


    Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.

  2. Glycosidase profiles of members of the family Enterobacteriaceae.

    PubMed Central

    Kämpfer, P; Rauhoff, O; Dott, W


    A total of 712 strains representing 47 taxa of the family Enterobacteriaceae were tested for the ability to hydrolyze 14 4-methylumbelliferyl (4-MU)-linked substrates within 3 h of incubation. In addition to the well-known differentiation potential of the hydrolysis of 4-MU-beta-D-galactopyranoside, 4-MU-beta-D-glucuronide, and 4-MU-beta-D-xylopyranoside, the hydrolysis of some other fluorogenic substrates (e.g., 4-MU-beta-D-fucopyranoside, 4-MU-N-acetyl-beta-D-galactosaminide, and 4-MU-alpha-D-galactopyranoside) can also be used for species differentiation within the family Enterobacteriaceae. PMID:1757564

  3. 7-O-Methylated anthocyanidin glycosides from Catharanthus roseus.


    Toki, Kenjiro; Saito, Norio; Irie, Yuki; Tatsuzawa, Fumi; Shigihara, Atsushi; Honda, Toshio


    Anthocyanins were isolated from orange-red flowers of Catharanthus roseus cv 'Equator Deep Apricot', and identified as rosinidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] (1), and also 7-O-methylcyanidin 3-O-[6-O-(alpha-rhamnopyranosyl)-beta-galactopyranoside] (2) by chemical and spectroscopic methods. Pigment 1 was found to be a major anthocyanin in the flowers of this cultivar. By contrast, the distribution of rosinidin glycosides is very limited in plants, and reported only in the flowers of Primula. Pigment 2 was found in smaller concentrations, but its aglycone, 7-O-methylcyanidin, has been reported only once before, from the fruit of mango.

  4. Convergent synthesis of a trisaccharide as its 2-(trimethylsilyl)ethyl glycoside related to the flavonoid triglycoside from Gymnema sylvestre.


    Mukhopadhyay, Balaram; Field, Robert A


    The glycone part of the flavonoid triglycoside, kaempferol 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside, has been synthesized in good yield and stereoselectivity using N-iodosuccinimide and HClO4-silica promoted glycosylations of thioglycoside donors.


    EPA Science Inventory

    A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the b...

  6. A new C-methylated flavonoid glycoside from Pinus densiflora.


    Jung, M J; Choi, J H; Chung, H Y; Jung, J H; Choi, J S


    A new C-methyl flavonol glycoside, 5,7,8,4'-tetrahydroxy-3-methoxy-6-methylflavone 8-O-beta-D-glucopyranoside (1), has been isolated from the needles of Pinus densiflora, together with kaempferol 3-O-beta-(6"-acetyl)-galactopyranoside.

  7. Septicaemia due to Neisseria lactamica--initial confusion with Neisseria meningitidis.


    Brown, N M; Ragge, N K; Speller, D C


    Neisseria lactamica, isolated from a baby with septicaemia, was at first thought to be Neisseria meningitidis, possibly acquired in hospital. Extensive investigation of contacts was made until the O-nitrophenyl-D-galactopyranoside reaction proved positive. Distinction between the two species, easily made in this way, is important both in individual patients and in population surveys.

  8. Reactions of immunoglobulin G-binding ligands with platelets and platelet-associated immunoglobulin G.

    PubMed Central

    Rosse, W F; Devine, D V; Ware, R


    Immunoglobulin G (IgG) bound to platelets is usually detected by one of two general methods: binding of labeled anti-IgG or consumption of anti-IgG. The latter method gives, in general, values 5-10-fold greater than the former under the same conditions. To investigate these discrepancies, we have compared the detection of platelet-bound IgG by a labeled anti-IgG binding assay and by a quantitative antiglobulin consumption test using the same antibodies. The interaction of 125I-labeled monoclonal anti-IgG or polyclonal anti-IgG with washed and IgG-coated platelets was studied. The binding of these ligands to washed normal platelets was largely (50-80%) nonspecific; the binding was not saturable and was only partially inhibitable by excess unlabeled anti-IgG. The binding of anti-IgG to platelets coated with anti-PIA1, a platelet-specific IgG antibody, appeared to be saturable and inhibitable; the dissociation constant (KD) of this IgG-anti-IgG reaction was 4.9 X 10(-9) for monoclonal and 1.4 X 10(-7) for polyclonal anti-IgG. The ratio of sites present on the membrane (determined by 131I-labeled anti-PIA1) to the number of binding sites for anti-IgG determined by Scatchard analysis was 0.53 for monoclonal anti-IgG and 1.3 for polyclonal anti-IgG. The binding of monoclonal anti-IgG to platelet-bound immune complexes or IgG aggregates appeared to be complex. 131I-Labeled IgG was affixed to platelets and was detected by three tests: direct binding of radiolabeled monoclonal anti-IgG and quantitative antiglobulin consumption (QAC) tests, which were quantitated either by measuring directly the amount of radiolabeled anti-IgG consumed from fluid phase (direct QAC), or indirectly by reference to a calibration curve relating the consumption of anti-IgG by known amounts of fluid-phase, non-immune IgG (indirect QAC). The amount of platelet-bound IgG detected by the direct binding of 125I-labeled monoclonal anti-IgG and by the direct QAC approximated that known to be bound to

  9. Recombinant anti-tenascin antibody constructs

    SciTech Connect



    The general objective of this research is to combine genetically derived molecular constructs reactive with tenascin, with appropriate radionuclides and labeling methods in order to generate more effective diagnostic and therapeutic reagents for oncologic nuclear medicine. Tenascin, a polymorphic extracellular matrix glycoprotein, is of interest because of its high expression on glioma, melanoma, as well as prostate and breast carcinoma. Recently, we have also documented high levels of tenascin in lymphomas, particularly those of higher grade, making the potential clinical impact of tenascin-specific radiodiagnostics and therapeutics even greater. An essential feature of our work plan is the ability to exploit our extensive clinical experience in order to design second-generation constructs with properties which could improve clinical efficacy. To date, we have treated over 150 brain tumor patients with 131I-labeled murine 81C6, an antibody which binds specifically to the alternatively spliced fibronectin type III repeats CD of the tenascin molecule. During the current grant period, we have made several observations which form the basis for our proposed specific aims. First, tissue distribution and catabolism experiments in animal models have demonstrated enhanced stability for a chimeric construct composed of murine variable regions and human IgG2 constant domains. Furthermore, pharmacokinetic studies in patients with 131I-labeled chimeric 81C6 have shown significantly longer retention in glioma tumor resection cavities compared with its murine parent. Second, we have initiated the first clinical trial of an endoradiotherapeutic labeled with the 7.2-hr -particle emitter 211At. Twelve glioma patients have received 211At-labeled chimeric 81C6 directly into their brain tumor resection cavity, and very encouraging results have been obtained. Now that the feasibility of human studies with 211At, has been demonstrated, the development and evaluation of anti

  10. Targeting radioimmunotherapy of hepatocellular carcinoma with iodine ({sup 131}I) metuximab injection: Clinical Phase I/II trials

    SciTech Connect

    Chen Zhinan . E-mail:; Mi Li; Xu Jing


    Purpose: HAb18G/CD147 is a hepatocellular carcinoma (HCC)-associated antigen. We developed iodine ({sup 131}I) metuximab injection (Licartin), a novel {sup 131}I-labeled HAb18G/CD147-specific monoclonal antibody F(ab'){sub 2} fragment, and evaluated its safety, pharmacokinetics, and clinical efficacy on HCC in Phase I/II trials. Methods and Materials: In a Phase I trial, 28 patients were randomly assigned to receive the injection in 9.25-, 18.5-, 27.75-, or 37-MBq/kg doses by hepatic artery infusion. In a multicenter Phase II trial, 106 patients received the injection (27.75 MBq/kg) on Day 1 of a 28-day cycle. Response rate and survival rate were the endpoints. Results: No life-threatening toxic effects were found. The safe dosage was 27.75 MBq/kg. The blood clearance fitted a biphasic model, and its half-life was 90.56-63.93 h. In the Phase II trial, the injection was found to be targeted and concentrated to tumor tissues. Of the 73 patients completing two cycles, 6 (8.22%) had a partial response, 14 (19.18%) minor response, and 43 (58.90%) stable disease. The 21-month survival rate was 44.54%. The survival rate of progression-free patients was significantly higher than that of patients with progressive disease after either one or two cycles (p < 0.0001 or p 0.0019). Conclusion: Iodine ({sup 131}I) metuximab injection is safe and active for HCC patients.

  11. Patient-specific dosimetry using quantitative SPECT imaging and three-dimensional discrete fourier transform convolution

    SciTech Connect

    Akabani, G.; Hawkins, W.G.; Eckblade, M.B.; Leichner, P.K.


    The objective of this study was to develop a three-dimensional discrete Fourier transform (3D-DFT) convolution method to perform the dosimetry for {sup 131}I-labeled antibodies in soft tissues. Mathematical and physical phantoms were used to compare 3D-DFT with Monte Carlo transport (MCT) calculations based on the EGS4 code. The mathematical and physical phantoms consisted of a sphere and cylinder, respectively, containing uniform and nonuniform activity distributions. Quantitative SPECT reconstruction was carried out using the circular harmonic transform (CHT) algorithm. The radial dose profile obtained from MCT calculations and the 3D-DFT convolution method for the mathematical phantom were in close agreement. The root mean square error (RMSE) for the two methods was <0.1%, with a maximum difference <21%. Results obtained for the physical phantom gave a RMSE <0.1% and a maximum difference of <13%; isodose contours were in good agreement. SPECT data for two patients who had undergone {sup 131}I radioimmunotherapy (RIT) were used to compare absorbed-dose rates and isodose rate contours with the two methods of calculations. This yielded a RMSE <0.02% and a maximum difference of <13%. Our results showed that the 3D-DFT convolution method compared well with MCT calculations. The 3D-DFT approach is computationally much more efficient and, hence, the method of choice. This method is patient-specific and applicable to the dosimetry of soft-tissue tumors and normal organs. It can be implemented on personal computers. 22 refs., 6 figs., 2 tabs.

  12. Review of five consecutive studies of radiolabeled immunoglobulin therapy in Hodgkin's disease.


    Vriesendorp, H M; Morton, J D; Quadri, S M


    Recurrent Hodgkin's Disease (HD) provides unique opportunities to improve radiolabeled immunoglobulin therapy (RIT). Normal tissue toxicity after RIT is limited to bone marrow damage and is well documented and quantified in HD patients. Anti-antibody formation is rare in patients with HD, allowing for multiple RIT cycles. Overall, 134 patients with recurrent HD were treated on five different studies with i.v. antiferritin, labeled with 131I or with 111In for diagnostic purposes and 90Y for therapeutic purposes. Patients with recurrent, end-stage HD obtain a 60% response rate following 90Y-labeled antiferritin. One-half of the therapy responses are complete. Responses are more common in patients with longer disease histories (> 3 years) and smaller tumor volumes (< 30 cm3) and in patients receiving at least 0.4 mCi 90Y-labeled antiferritin/kg body weight. Complete responders survive significantly longer than partial responders (2 years versus 1 year). Partial responders survive longer than patients with progressive disease (1 year versus 4 months). HD in one-third of the patients recurs in new areas. A low protein dose (2-5 mg) and a moderate specific activity (10 mCi/mg) are recommended. Results obtained with 90Y-labeled antiferritin are significantly better than results with 131I-labeled antiferritin. Further translational research in vitro in the radio pharmacy and in vivo with experimental animals is ongoing to improve the therapeutic results of RIT in HD. Obviously, many permutations of RIT cannot be explored in HD patients for ethical, financial, or logistic reasons, and predictive preclinical research is required to achieve further progress. Currently, RIT is a low-toxicity, low-cost outpatient procedure for recurrent HD with a high response rate in a patient population with an unfavorable prognosis.

  13. CT-SPECT fusion plus conjugate views for determining dosimetry in iodine-131-monoclonal antibody therapy of lymphoma patients

    SciTech Connect

    Koral, K.F.; Zasadny, K.R.; Kessler, M.L.


    A method of performing {sup 131}I quantitative SPECT imaging is described which uses the superimposition of markers placed on the skin to accomplish fusion of computed tomography (CT) and SPECT image sets. To calculate mean absorbed dose after administration of one of two {sup 131}I-labeled monoclonal antibodies (Mabs), the shape of the time-activity curve is measured by daily diagnostic conjugate views, the y-axis of that curve is normalized by a quantitative SPECT measurement (usually intra-therapy), and the tumor mass is deduced from a concurrent CT volume measurement. The method is applied to six B-cell non-Hodgkin`s lymphoma patients. For four tumors in three patients treated with the MB1 Mab, a correlation appears to be present between resulting mean absorbed dose and disease response. Including all dosimetric estimates for both antibodies, the range for the specific absorbed dose is within that found by others in treating B-cell lymphoma patients. Excluding a retreated anti-B1 patient, the tumor-specific absorbed dose during anti-B1 therapy is from 1.4 to 1.7 mGy/MBq. For the one anti-B1 patient, where quantitative SPECT and conjugate-view imaging was carried out back to back , the quantitative SPECT-measured activity was somewhat less for the spleen and much less for the tumor than that from conjugate views. The quantitative SPECT plus conjugate views method may be of general utility for macro-dosimetry of {sup 131}If therapies. 18 refs., 3 figs., 5 tabs.

  14. Radioimmunotherapy: Development of an effective approach

    SciTech Connect

    DeNardo, S.J.


    We plan to extend our success in treating B cell malignancies with {sup 131}I labeled Lym-1 by a major effort in therapy with {sup 67}Cu Lym-1. Yttrium-90 labeled by a macrocycle, DOTA will be studied in patients as a continuation of the {sup 111}In-BAD (DOTA) Lym-1 studies. Excellent images and pharmacokinetics of the {sup 111}In-BAD(DOTA)-Lym-1 studies. Lymphomas and related diseases represent a special case for radioimmunotherapy because of their documented radiosensitivity and immunodeficiency, and thus offer a unique opportunity to conduct therapeutic feasibility studies in a responsive human model. Using marine and chimeric L6 and other MoAb to breast cancer, we have applied the strategies that were developed in taking Lym-1 antibody from the bench to the patient. We have examined a number of monoclonal antibodies for treatment of breast cancer and chose chimeric L6 for prototype studies because of certain characteristics. The chemistry of attachment of conjugates to antibodies and their impact on immunological targeting biological activities (cytotoxicity), metabolic fate, and therapeutic index will continue to be a major strength and function of this program. This grant has supported the conception, synthesis, and development of the first macrocylic, bifunctional chelating agent TETA (6-p-nitrobenzyl-1,4,8,11-tetraazatetradecane-N,N{prime},N{double prime}, N{prime}{double prime}-tetraacetic acid and its derivatives, including Lym-1-2IT-BAT), for use in Cu-67-based radioimmunodiagnosis and therapy. This work has led to the further development of several new macrocylic bifunctional chelating agents for copper, indium, yttrium and other metals. In addition, successful Cu-67 labelings of Lym-1-2IT-BAT for human radiopharmaceutical have shown patient pharmacokinetics of {sup 67}Cu-BAT(TETA)-Lym-1 with promising therapeutic dosimetry.

  15. Radioimmunotherapy: Development of an effective approach. Annual report, 1991

    SciTech Connect

    DeNardo, S.J.


    We plan to extend our success in treating B cell malignancies with {sup 131}I labeled Lym-1 by a major effort in therapy with {sup 67}Cu Lym-1. Yttrium-90 labeled by a macrocycle, DOTA will be studied in patients as a continuation of the {sup 111}In-BAD (DOTA) Lym-1 studies. Excellent images and pharmacokinetics of the {sup 111}In-BAD(DOTA)-Lym-1 studies. Lymphomas and related diseases represent a special case for radioimmunotherapy because of their documented radiosensitivity and immunodeficiency, and thus offer a unique opportunity to conduct therapeutic feasibility studies in a responsive human model. Using marine and chimeric L6 and other MoAb to breast cancer, we have applied the strategies that were developed in taking Lym-1 antibody from the bench to the patient. We have examined a number of monoclonal antibodies for treatment of breast cancer and chose chimeric L6 for prototype studies because of certain characteristics. The chemistry of attachment of conjugates to antibodies and their impact on immunological targeting biological activities (cytotoxicity), metabolic fate, and therapeutic index will continue to be a major strength and function of this program. This grant has supported the conception, synthesis, and development of the first macrocylic, bifunctional chelating agent TETA (6-p-nitrobenzyl-1,4,8,11-tetraazatetradecane-N,N{prime},N{double_prime}, N{prime}{double_prime}-tetraacetic acid and its derivatives, including Lym-1-2IT-BAT), for use in Cu-67-based radioimmunodiagnosis and therapy. This work has led to the further development of several new macrocylic bifunctional chelating agents for copper, indium, yttrium and other metals. In addition, successful Cu-67 labelings of Lym-1-2IT-BAT for human radiopharmaceutical have shown patient pharmacokinetics of {sup 67}Cu-BAT(TETA)-Lym-1 with promising therapeutic dosimetry.

  16. Improved therapeutic outcomes of thermal ablation on rat orthotopic liver allograft sarcoma models by radioiodinated hypericin induced necrosis targeted radiotherapy

    PubMed Central

    Gao, Long; Zhang, Jian; Ma, Tengchuang; Yao, Nan; Gao, Meng; Shan, Xin; Ni, Yicheng; Shao, Haibo; Xu, Ke


    Residual tumor resulting in tumor recurrence after various anticancer therapies is an unmet challenge in current clinical oncology. This study aimed to investigate the hypothesis that radioiodinated hypericin (131I-Hyp) may inhibit residual tumor recurrence after microwave ablation (MWA) on rat orthotopic liver allograft sarcoma models. Thirty Sprague-Dawley (SD) rats with hepatic tumors were divided into three groups: Group A received laparotomy MWA and sequential intravenous injection (i.v.) of 131I labelled hypericin (131I-Hyp) in a time interval of 24 h; Group B received only laparotomy MWA; Group C was a blank control. Tumor inhibitory effects were monitored with in vivo magnetic resonance imaging (MRI) and these findings were compared to histopathology data before (baseline, day 0) and 1, 4, and 8 days after MWA. In addition, biodistribution of 131I-Hyp was assessed with in vivo single-photon emission computed tomography-computed tomography (SPECT-CT) imaging, in vitro autoradiography, fluorescent microscopy, and gamma counting. A fast clearance of 131I-Hyp and increasing deposit in necrotic tumors appeared over time, with a significantly higher radioactivity than other organs (0.9169 ± 1.1138 % ID/g, P < 0.01) on day 9. Tumor growth was significantly slowed down in group A compared to group B and C according to MRI images and corresponding tumor doubling time (12.13 ± 1.99, 4.09 ± 0.97, 3.36 ± 0.72 days respectively). The crescent tagerability of 131I-Hyp to necrosis was visualized consistently by autoradiography and fluorescence microscopy. In conclusion, 131I-Hyp induced necrosis targeted radiotherapy improved therapeutic outcomes of MWA on rat orthotopic liver allograft sarcoma models. PMID:27285983

  17. Anti-cancer activity of curcumin loaded nanoparticles in prostate cancer

    PubMed Central

    Yallapu, Murali M.; Khan, Sheema; Maher, Diane M.; Ebeling, Mara C.; Sundram, Vasudha; Chauhan, Neeraj; Ganju, Aditya; Balakrishna, Swati; Gupta, Brij K.; Zafar, Nadeem; Jaggi, Meena; Chauhan, Subhash C.


    Prostate cancer is the most commonly diagnosed cancer disease in men in the Unites States and its management remains challenge in everyday oncology practice. Thus, advanced therapeutic strategies are required to treat prostate cancer patients. Curcumin (CUR) is a promising anticancer agent for various cancer types. The objective of this study was to evaluate therapeutic potential of novel poly(lactic-co-glycolic acid)- CUR nanoparticles (PLGA-CUR NPs) for prostate cancer treatment. Our results indicate that PLGA-CUR NPs efficiently internalize in prostate cancer cells and release biologically active CUR in cytosolic compartment of cells for effective therapeutic activity. Cell proliferation (MTS), clonogenic, and Western blot analyses reveal that PLGA-CUR NPs can effectively inhibit proliferation and colony formation ability of prostate cancer cells than free CUR. PLGA-CUR NPs showed superior tumor regression compared to CUR in xenograft mice. Further investigations reveal that PLGA-CUR NPs inhibit nuclear β-catenin and AR expression in cells and in tumor xenograft tissues. It also suppresses STAT3 and AKT phosphorylation and leads to apoptosis via inhibition of key anti-apoptotic proteins, MCL-1, Bcl-xL and caused induction of PARP cleavage. Additionally, PLGA-CUR NPs significant downregulation of oncogenic miR21 and up-regulation of miR-205 was observed with PLGA-CUR NPs treatment as determined by RT-PCR and in situ hybridization analyses. A superior anti-cancer potential was attained with PSMA antibody conjugated PLGA-CUR NPs in prostate cancer cells and a significant tumor targeting of 131I labelled PSMA antibody was achieved with PLGA-CUR NPs in prostate cancer xenograft mice model. In conclusion, PLGA-CUR NPs can significantly accumulate and exhibit superior anticancer activity in prostate cancer. PMID:25028336

  18. Albumin modulates lateral assembly of fibrin polymers: evidence of enhanced fine fibril formation and of unique synergism with fibrinogen

    SciTech Connect

    Galanakis, D.K.; Lane, B.P.; Simon, S.R.


    The authors identified a new property of human albumin. It enhances formation of fine fibril (or leptofibril) structure during fibrin gelation, and by nephelometric and electron microscopic measurements, this property is independent of and synergistic with that of fibrinogen. They examined fibrin aggregation using physiologic temperatures and pH and albumin:fibrin concentration ratios below those at which the known accelerating effect on fibrin aggregation occurs. An albumin concentration dependent decrease in gel turbidity maxima was consistently demonstrable in buffers containing or lacking (2-5 mM) CaCl/sub 2/. Electron microscopic measurements of cross-sectional fibril widths, performed on sections of glutaraldehyde-fixed gels, disclosed differences between albumin-containing and control gels which were significant. Spin-labeled albumin displayed no change in electron (para) magnetic spin resonance spectral measurements during its inhibition of fibrin, indicating no perturbation on albumin conformation in the vicinities of Cys-34 and of fatty acid binding sites. Certain fibrinogen:albumin ratios designed to induce maximal inhibition yet permit gelation in the presence of either alone prevented gelation of buffer-diluted fibrin monomers. Aliquots from these which were dried and negatively stained on formvar-coated grids disclosed strands of 5-17 nm width, most displaying a 60-250-nm approximate length. The amounts of /sup 131/I-labeled coagulable fibrin which remained soluble in fibrinogen solutions were increased by albumin. They conclude that albumin enhances formation of leptofibril-rich gel domains when other plasma factors favor formation of such structures. Available evidence indicating decreased permeability implies that such gel domains limit efflux rates from the intrathrombus environment and from intra- to extravascular space.

  19. Imaging of brain tumor proliferative activity with iodine-131-iododeoxyuridine

    SciTech Connect

    Tjuvajev, J.G.; Macapinlac, H.A.; Daghighian, F.


    Iodine-131-iododeoxyuridine (IUdR) uptake and retention was imaged with SPECT at 2 and 24 hr after administering a 10-mCi dose to six patients with primary brain tumors. The SPECT images were directly compared to gadolinium contrast-enhanced MR images as well as to ({sup 18}F) fluorodeoxyglucose (FDG) PET scans and {sup 201}Tl SPECT scans. Localized uptake and retention of IUdR-derived radioactivity was observed in five of six patients. The plasma half-life of ({sup 131}I) IUdR was short (1.5 min) in comparison to the half-life of total plasma radioactivity (6.4 hr). The pattern of ({sup 131}I)IUdR-derived radioactivity was markedly different in the 2-hr compared to 24-hr images. Radioactivity was localized along the periphery of the tumor and extended beyond the margin of tumor identified by contrast enhancement on MRI. The estimated levels of tumor radioactivity at 24 hr, based on semiquantitative phantom studies, ranged between <0.1 and 0.2 {mu}Ci/cc (<0.001% and 0.002% dose/cc); brain levels were not measurable. Iodine-131-IUdR SPECT imaging of brain tumor proliferation has low (marginal) sensitivity due to low count rates and can detect only the most active regions of tumor growth. Imaging at 24 hr represents a washout strategy to reduce {sup 131}I-labeled metabolites contributing to background activity in the tumors, and is more likely to show the pattern of ({sup 131}I)IUdR-DNA incorporation and thereby increase image specificity. Iodine-123-IUdR SPECT imaging at 12 hr and the use of ({sup 124}I)IUdR and PET will improve count acquisitions and image quality. 74 refs., 6 figs., 2 tabs.

  20. 131I-Anti-CD45 Antibody Plus Busulfan and Cyclophosphamide before Allogeneic Hematophoietic Cell Transplantation for Treatment of Acute Myeloid Leukemia in First Remission

    SciTech Connect

    Pagel, John M.; Appelbaum, Frederick R.; Eary, Janet F.; Rajendran, Joseph G.; Fisher, Darrell R.; Gooley, Ted; Ruffner, Katherine; Nemecek, Eneida; Sickle, Eileen; Durack, Larry; Carreras, Jeanette; Horowitz, Mary; Press, Oliver W.; Gopal, Ajay K.; Martin, Paul J.; Bernstein, Irwin D.; Matthews, Dana C.


    In an attempt to improve outcomes for patients with acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (HCT), we conducted a Phase I/II study in which targeted irradiation delivered by 131I-anti-CD45 antibody was combined with targeted busulfan (BU; area-under-curve, 600-900 ng/ml) and cyclophosphamide (CY; 120 mg/kg). Fifty-two of 59 patients (88%) receiving a trace 131I-labeled dose of 0.5 mg/kg anti-CD45 murine antibody had higher estimated absorbed radiation in bone marrow and spleen than in any other organ. Forty-six patients were treated with 102-298 mCi 131I delivering an estimated 5.3-19 (mean 11.3) Gy to marrow, 17-72 (mean 29.7) Gy to spleen, and 3.5 Gy (n=4) to 5.25 Gy (n=42) to the liver. The estimated 3-year non-relapse mortality and disease-free survival (DFS) were 21% and 61%, respectively. These results were compared to those from 509 similar International Bone Marrow Transplant Registry patients transplanted using BU/CY alone. After adjusting for differences in age and cytogenetics-risk, the hazard of mortality among all antibody-treated patients was 0.65 times that of the Registry patients (95% CI 0.39-1.08; p=.09). The addition of targeted hematopoietic irradiation to conventional BU/CY is feasible and well tolerated, and Phase II results are sufficiently encouraging to warrant further study.

  1. Conditionally cleavable radioimmunoconjugates: a novel approach for the release of radioisotopes from radioimmunoconjugates.


    Beeson, Craig; Butrynski, James E; Hart, Michael J; Nourigat, Cynthia; Matthews, Dana C; Press, Oliver W; Senter, Peter D; Bernstein, Irwin D


    One of the limitations of therapy with radiolabeled monoclonal antibodies (mAbs) is that significant toxicities can arise from circulating non-tumor-bound radiolabeled conjugate. Here, we describe a new method to reduce systemic radiation exposure from radiolabeled mAbs involving the attachment of the radioisotope through a linker that can be cleaved by an administered enzyme. To demonstrate the feasibility of this approach, we prepared a conditionally cleavable radioimmunoconjugate (RIC) composed of (131)I-labeled cephalosporin conjugated to Tositumomab, a mAb against the CD20 antigen. The cleavable RIC bound antigen identically to directly iodinated antibody, and in the presence of beta-lactamase, about 80-85% of the radioisotope was released. In vivo studies in mice revealed that the cleavable RIC and the directly iodinated anti-CD20 antibody had similar biodistribution patterns. Systemically administered beta-lactamase induced a 2-3-fold decrease in the percent injected dose (ID) of the cleavable RIC/g of blood, marrow, spleen, lung, and liver 1 h after enzyme treatment, and a 4-6-fold decrease 20 h after enzyme treatment. This was accompanied by a 20-fold increase in % ID/g in urine 1 h after enzyme treatment, indicating that the released radiolabel was rapidly excreted through the kidneys. In mice with human tumor xenografts, there was no decrease in the %ID/g in tumor 1 h after enzyme treatment, but by 4 h after enzyme injection, decreases in tumor radioactive content began to diminish the targeting advantage. These studies demonstrate that the cleavable RIC substrate is able to bind to tumor antigens and localize within human tumor xenografts and that accelerated systemic clearance can be induced with beta-lactamase.

  2. Modulation of hemodynamic and vascular filtration changes in diabetic rats by dietary myo-inositol

    SciTech Connect

    Pugliese, G.; Tilton, R.G.; Speedy, A.; Santarelli, E.; Eades, D.M.; Province, M.A.; Kilo, C.; Sherman, W.R.; Williamson, J.R. )


    To assess the potential of myo-inositol-supplemented diets to prevent diabetes-induced vascular functional changes, we examined the effects of diets supplemented with 0.5, 1, or 2% myo-inositol on blood flow and vascular filtration function in nondiabetic control rats and rats with streptozocin-induced diabetes (STZ-D). After 1 mo of diabetes and dietary myo-inositol supplementation, (1) 131I-labeled bovine serum albumin (BSA) permeation of vessels was assessed in multiple tissues, (2) glomerular filtration rate (GFR) was estimated as renal plasma clearance of 57Co-labeled EDTA, (3) regional blood flows were measured with 15-microns 85Sr-labeled microspheres, and (4) endogenous albumin and IgG urinary excretion rates were quantified by radial immunodiffusion assay. In STZ-D rats, 131I-BSA tissue clearance increased significantly (2- to 4-fold) in the anterior uvea, choroid-sclera, retina, sciatic nerve, aorta, new granulation tissue, diaphragm, and kidney but was unchanged in skin, forelimb muscle, and heart. myo-Inositol-supplemented diets reduced diabetes-induced increases in 131I-BSA clearance (in a dose-dependent manner) in all tissues; however, only in new granulation tissue and diaphragm did the 2% myo-inositol diet completely normalize vascular albumin permeation. Diabetes-induced increases in GFR and in urinary albumin and IgG excretion were also substantially reduced or normalized by dietary myo-inositol supplements. Increased blood flow in anterior uvea, choroid-sclera, kidney, new granulation tissue, and skeletal muscle in STZ-D rats also was substantially reduced or normalized by the 2% myo-inositol diet. myo-Inositol had minimal if any effects on the above parameters in control rats.

  3. Radioimmunodetection of colorectal cancer

    SciTech Connect

    Kim, E.E.; Deland, F.H.; Casper, S.; Corgan, R.L.; Primus, F.J.; Goldenberg, D.M.


    This study examines the accuracy of colorectal cancer radioimmunodetection. Twenty-seven patients with a history of histologically-confirmed colonic or rectal carcinoma received a high-titer, purified goat anti-CEA IgG labelled with /sup 131/I at a total dose of at least 1.0 Various body views were scanned at 24 and 48 hours after administration of the radioantibody. Three additional cases were evaluated; one had a villous adenoma in the rectum and received the /sup 131/I-labeled anti-CEA IgG, while two colonic carcinoma patients received normal goat IgG labelled with /sup 131/I. All of the 7 cases with primary colorectal cancer showed true-positive tumor localization, while 20 of 25 sites of metastatic colorectal cancer detected by immune scintigraphy were corroborated by other detection measures. The sensitivity of the radioimmunodetection of colorectal cancers (primary and metastatic) was found to be 90% (true-positive rate), the putative specificity (true-negative rate) was 94%, and the apparent overall accuracy of the technique was 93%. Neither the case of a villous adenoma receiving the anti-CEA IgG nor the two cases of colonic cancer receiving normal goat IgG showed tumor radiolocalization. Very high circulating CEA titers did not appear to hinder successful tumor radiolocalization. These findings suggest that in colorectal cancers the method of CEA radioimmunodetection may be of value in preoperatively determining the location and extent of disease, in assessing possible recurrence or spread postoperatively, and in localizing the source of CEA production in patients with rising or elevated CEA titers. An ancilliary benefit could be a more tumor-specific detection test for confirming the findings of other, more conventional diagnostic measures.

  4. Multi-modality treatment of primary nonresectable intrahepatic cholangiocarcinoma with /sup 131/I anti-CEA--a Radiation Therapy Oncology Group Study

    SciTech Connect

    Stillwagon, G.B.; Order, S.E.; Klein, J.L.; Leichner, P.K.; Leibel, S.A.; Siegelman, S.S.; Fishman, E.K.; Ettinger, D.S.; Haulk, T.; Kopher, K.


    Thirty-seven patients with primary nonresectable intrahepatic cholangiocarcinoma (57% with prior treatment and/or metastasis) were prospectively treated with external radiation, chemotherapy, and /sup 131/I labelled anti-CEA. Therapy began in all trials with whole liver irradiation (21.0 Gy, 3.0 Gy/Fx, 4 days/week, 10 MV photons) with alternate treatment day chemotherapy (Adriamycin, 15 mg + 5-FU, 500 mg). One month after external beam therapy, chemotherapy was given (Adriamycin, 15 mg + 5-FU, 500 mg) followed the next day by the first administration of /sup 131/I anti-CEA. The treatment schedule used was 20 mCi day 0; 10 mCi day 5 as an outpatient. This schedule was derived from tumor dose estimates which indicated that 20 mCi (8-10 mCi/mg IgG) was sufficient to achieve tumor saturation with a tumor effective half-life of 3 to 5 days, depending upon the species of animal from which the antibody was obtained. The median tumor dose for the 20 mCi + 10 mCi regimen was 6.2 Gy. Antibody therapy was delivered in 2-month cycles using antibody generated in different species of animals; rabbit, pig, monkey, and bovine. Toxicity was limited to hematologic toxicity and was manifested as thrombocytopenia and leukocytopenia (3.2% Grade IV for each according to RTOG toxicity criteria). Tumor remission evaluated by CT scan digitized tumor volume analysis indicated a 26.6% partial response (PR). Tumor remission by physical examination indicated a 33.3% remission rate (25.9% PR and 7.4% complete remission (CR). The median survival for patients who responded was 15.2 months. The actuarial median survival for the entire group of patients (metastases and previous treatment) was 6.5 months. The longest partial remission is presently more than 4 years.

  5. Antibody distribution and dosimetry in patients receiving radiolabelled antibody therapy for colorectal cancer.

    PubMed Central

    Begent, R. H.; Ledermann, J. A.; Green, A. J.; Bagshawe, K. D.; Riggs, S. J.; Searle, F.; Keep, P. A.; Adam, T.; Dale, R. G.; Glaser, M. G.


    The distribution of iodine-131 (131I) labelled antibody to carcinoembryonic antigen (CEA) has been studied in 16 patients with colorectal cancer. Levels of tumour and normal tissue radioactivity were measured by serial gamma-camera imaging and counting of blood and urine. Maximum concentrations were found in tumour 8 h after administration and varied up to 9-fold in different patients. Higher levels were found on average in tumour than in any other tissue. Liver, lung and blood were the other tissues in which antibody was concentrated relative to the rest of the body. Antibody cleared from all these tissues over 1 week. Second antibody directed against the antitumour (first) antibody was given 24 h after first antibody in order to accelerate clearance from the blood. This increased the tumour to blood ratio but had little effect on other tissues. Cumulative radiation dose to tumour and normal tissue was estimated. In patients with the most efficient localisation the tumour to body ratio was 20:1 and tumour to blood ratio 5:1. This may be sufficient for effective therapy of cancer in patients selected for efficient antibody localisation. The data may be used to estimate the effect of different therapeutic strategies. For instance, in the time after second antibody administration the average tumour to blood ratio of radiation dose was 11:1, suggesting that two phase systems in which the therapeutic modality is given after a good tumour to normal tissue ratio is obtained may be effective for the majority of patients. Images Figure 2 Figure 4 PMID:2789951

  6. Interactions of calcium nitrate with pyranosides in water: A 13C NMR study

    NASA Astrophysics Data System (ADS)

    Zhuo, Kelei; Wang, Yaping; Zhao, Yang; Liu, Qian; Wang, Jianji


    The 13C NMR spectra of methyl α- and β- D-galactopyranosides, and methyl α- and β- D-glucopyranosides were recorded and show that the Δ( δC-4) values for methyl α- and β- D-galactopyranosides increase most rapidly, whereas those for methyl α- and β- D-glucopyranosides vary hardly with increasing molality of calcium nitrate. It can be concluded that ax-OH-4 interacts more strongly with Ca 2+ than eq-OH-4 group, namely, the Ca 2+ ion interaction with ax-OH-4 leads to a stronger deshielding of the C-4 atom. Compared with other C atoms, the chemical shifts of both C-1 and C-5 atoms in these two types of glycosides decrease relatively rapidly as molality of calcium nitrate increases, indicating that the nitrate ion attractions for these glycosides cause a relatively strong enhancing shielding effect of C-1 and C-5 atoms.

  7. The first report on flavonoid isolation from Annona crassiflora Mart.


    Lage, Gisele Avelar; Medeiros, Felipe da Silva; Furtado, Werônica de Lima; Takahashi, Jacqueline Aparecida; de Souza Filho, José Dias; Pimenta, Lúcia Pinheiro Santos


    Annona crassiflora, a native tree from Brazilian Cerrado, is reported to possess several ethnomedical uses. Here, we report the isolation and unambiguous characterisation of the flavonoids quercetin-3-O-β-D-glucopyranosil(1 → 6)-O-α-L-arabinoside (1), known as peltatoside, kaempferol-3-O-β-D-galactopyranoside (2), quercetin-3-O-β-D-galactopyranoside (3), quercetin-3-O-β-L-arabinopiranoside (4) and the ( - )-epicatechin (5) from the hydroalcoholic portion of the leaf ethanolic extract. Their structures were elucidated by using 1D and 2D NMR, ESI-MS, UV/Vis spectroscopy, optical rotation analysis and literature data comparison. The leaf ethanolic extract and its isolated compounds were evaluated by using antimicrobial, antioxidant and larvicidal assays, expressing antimicrobial and antioxidant activities. This is the first report on flavonoid isolation from A. crassiflora.

  8. Anthocyanins from the scarlet flowers of Anemone coronaria.


    Toki, K; Saito, N; Shigihara, A; Honda, T


    Three acylated anthocyanins were isolated from the scarlet flowers of Anemone coronaria 'St. Brigid Red' along with a known pigment, pelargonidin 3-lathyroside. The structures of the acylated pigments were based on a pelargonidin 3-lathyroside skeleton acylated at different positions with malonic acid. The first pigment was identified as pelargonidin 3-O-[2-(beta-D-xylopyranosyl)-6-O-(malonyl)-beta-D-galactopyranoside], the second was pelargonidin 3-O-[2-O-(beta-D-xylopyranosyl)-6-O-(methyl-malonyl)-beta-D-galactopyranoside], and the third was (6''-O-(pelargonidin 3-O-[2''-O-(beta-D-xylopyranosyl)-beta-D-galactopyranosyl]))((4-O-(beta-D-glucopyranosyl)-trans-caffeoyl)-O-tartatryl)malonate.

  9. Canna indica flower: New source of anthocyanins.


    Srivastava, Jyoti; Vankar, Padma S


    In this study the red flowers of Canna indica (Cannaceae) were extracted by using sonicator and isolation of anthocyanins have been carried out. Four anthocyanin pigments have been isolated apart from quercetin and lycopene. They are Cyanidin-3-O-(6''-O-α-rhamnopyranosyl)-β-glucopyranoside (1), Cyanidin-3-O-(6''-O-α-rhamnopyranosyl)-β-galactopyranoside (2), Cyanidin-3-O-β-glucopyranoside (3) and Cyanidin-O-β-galactopyranoside (4). These compounds were isolated by using HPLC and their structures were subsequently determined on the basis of spectroscopic analyses, i.e., (1)H NMR, (13)C NMR, HMQC, HMBC, ESI-MS, FTIR, UV-Visible etc. The isolated compounds showed good antioxidant activity thus makes it suitable for use in food coloration and as a nutraceutical. Thus it is a promising pigment source for food applications.

  10. Glycosylation by sugar 1,2-O-(1-cyanobenzylidene) derivatives: influence of glycosyl-donor structure and promoter.


    Kochetkov, N K; Betaneli, V I; Kryazhevskikh, I A; Ott AYa


    3,4,6-Tri-O-acetyl-1,2-O-(1-cyanobenzylidene)-alpha-D-galactopy ranoses with differently substituted aromatic nuclei have been synthesised and used as glycosyl donors in reactions with the acceptors methyl 2,3,4-tri-O-acetyl-6-O-trityl-beta-D-galactopyranoside (11) and methyl 2,4,6-tri-O-acetyl-3-O-trityl-beta-D-galactopyranoside (12). The glycosylation of 11 gave only beta products. The effeciency and stereoselectivity of the glycosylations of 12 depend on the nature of the substituent in the aromatic ring. The stereoselectivity for the formation of 1,2-trans-glycosidic bonds was the highest with the p-methoxy-benzylidene derivative with triphenylmethylium triflate as the promoter.

  11. Two new flavonol glycosides from Gymnema sylvestre and Euphorbia ebracteolata.


    Liu, Xin; Ye, Wencai; Yu, Biao; Zhao, Shouxun; Wu, Houming; Che, Chuntao


    Two new flavonol glycosides, namely kaempferol 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside (1) and quercetin 3-O-6"-(3-hydroxyl-3-methylglutaryl)-beta-D-glucopyranoside (2), have been isolated from the aerial parts of Gymnema sylvestre and Euphorbia ebracteolata, respectively. Their structures were determined on the basis of chemical and spectroscopic methods.

  12. Breast Cancer Gene Therapy: Development of Novel Non-Invasive Magnetic Resonance Assay to Optimize Efficacy

    DTIC Science & Technology


    2 Task 5 Scale up synthesis of most promising indicator for animal investigations (Months 13-15) (Completed in Year 2) Task 6 Evaluate in mice...remaining tasks will be completed successfully. Task 6 Completed during Year 3. We found that administration of PFONPG (4-Fluoro-2- nitrophenol -β-D...galactopyranoside) to ROSA mice caused rapid death. We believe this is due to release of toxic aglycon ( nitrophenol ) causing rapid depolarization of the

  13. Organogel-assisted topochemical synthesis of multivalent glyco-polymer for high-affinity lectin binding.


    Krishnan, Baiju P; Raghu, Sreedevi; Mukherjee, Somnath; Sureshan, Kana M


    An organogelator, 2,4-undeca-diynyl-4',6'-O-benzylidene-β-d-galactopyranoside, which aligns its diacetylene upon gelation, has been synthesized. UV irradiation of its gel resulted in topochemical polymerization of the gelator forming polydiacetylene (PDA). We have used this gel-state reaction for the synthesis of surface-immobilized multi-valent glycoclusters, which showed 1000-fold enhanced binding, compared to monomers, with various galactose-binding lectins.

  14. Triterpenoid glycosides from bark of Meliosma lanceolata.


    Abe, F; Yamauchi, T; Shibuya, H; Kitagawa, I


    From the bark of Meliosma lanceolate, nine triterpenoid glycosides including the 28-O-beta-D-glucopyranosyl ester of bayogenin-3-O-beta-D-glucuronopyranoside, its 4'-O-beta-D-galactopyranoside and 4'-O-alpha-L-arabinopyranoside were isolated and the structures characterized along with that of 28-O-beta-D-glucopyranosyl-bayogenin-3-O-beta-D-4'-anhydro-4', 5'-didehydroglucuronopyranoside. Bisdesmosidic triosides of hederagenin were obtained as minor components.

  15. Rapid Confirmation of Clostridium perfringens by Using Chromogenic and Fluorogenic Substrates

    PubMed Central

    Adcock, Philip W.; Saint, Christopher P.


    The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-β-d-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life. PMID:11526053

  16. Melibiose is hydrolyzed exocellularly by an inducible exo-alpha-galactosidase in Azotobacter vinelandii.


    Wong, T Y


    Azotobacter vinelandii hydrolyzed melibiose exocellularly, leading to an accumulation of free glucose and galactose in the medium. This enzyme could also be induced by galactose, raffinose, and stachyose. The alpha-galactosidase activity could be detected quantitatively by using p-nitrophenyl-alpha-galactopyranoside as a substrate for intact cells. Chloramphenicol totally inhibited the induction of this enzyme. However, benzyl alcohol inhibited the secretion of this enzyme but did not inhibit the biosynthesis of the enzyme.

  17. Rapid microbiochemical method for presumptive identification of gastroenteritis-associated members of the family Enterobacteriaceae.


    Yong, D C; Thompson, J S; Prytula, A


    A method for rapid screening of isolates of pathogenic members of the family Enterobacteriaceae is described. Flow charts are used in conjunction with triple sugar iron agar, o-nitrophenyl-beta-D-galactopyranoside-phenylalanine-motility sulfate screening media, oxidase test, and six rapid biochemical tests, namely, lysine decarboxylase, urease, indole, esculin hydrolysis, malonate, and xylose. This scheme is used to provide an inexpensive but rapid presumptive identification of Salmonella, Shigella, Edwardsiella, Aeromonas, Plesiomonas, Vibrio, and Yersinia isolates from stool cultures.

  18. [Flavonoids from Humulus lupulus].


    Zhang, Wei-Ku; Wang, Shou-Bao; Fu, Cheng-Yu; Li, Ping; Xu, Jie-Kun


    Nine compounds were isolated and purified by column chromatographic techniques including macroporous resin, silica gel, ODS, Sephadex LH-20, and preparative reversed-phase HPLC. Their structures were elucidated as taxifolin (1), naringenin (2), chalconaringenin (3), acacetin (4), quercetin 3-O-beta-D-galactopyranoside (5), 6-prenylnaringenin (6) xanthohumol (7), desmethylxanthohumol (8), xanthohumol B (9) on the basis of MS and NMR spectroscopic data analysis. Compounds 1-5 were isolated from Humulus lupulus for the first time.

  19. Structure-function relations of carbohydrates by neoglycolipid arrays.


    Huang, Gang-Liang; Huang, Hua-Liang; Zhang, Hou-Cheng; Wang, Peng-George


    The work presented herein is a new noncovalent glycoarray assembly method for microplates created by simply mixing together a carbohydrate and a tetradecylamine. alpha-D-Mannopyranoside, alpha-D-glucopyranoside, and alpha-D-galactopyranoside were utilized in model studies and product formations were detected by lectin binding. The method can be extended to study the steric hindrance effect of carbohydrate-protein interactions, namely the structure-function relations of carbohydrates.

  20. Catalytic and regioselective oxidation of carbohydrates to synthesize keto-sugars under mild conditions.


    Muramatsu, Wataru


    A new catalytic and regioselective approach for the synthesis of keto-sugars is described. An organotin catalyst, Oc2SnCl2, in the presence of trimethylphenylammonium tribromide ([TMPhA](+)Br3(-)) accelerates the regioselective oxidation at the "axial"-OH group of 1,2-diol moieties in galactopyranosides. The reaction conditions can also be used for the regioselective oxidation of various carbohydrates.

  1. Anthocyanins from red flower tea (Benibana-cha), Camellia sinensis.


    Terahara, N; Takeda, Y; Nesumi, A; Honda, T


    Three anthocyanins were isolated from the leaves of red flower tea (Benibana-cha), Camellia sinensis, and their structures were determined by means of chemical and spectroscopic analyses. Two are the anthocyanins, delphinidin and cyanidin 3-O-beta-D-galactosides, respectively. Whereas the third, delphinidin 3-0-beta-D-(6-(E)-p-coumaryl)galactopyranoside. The anthocyanins were also contained in the flowers of Benibana-cha in different compositions.

  2. A tigogenin pentasaccharide from Cestrum diurnum.


    Ahmad, V U; Baqai, F T; Ahmad, R


    A new steroidal saponin named diurnoside 1 has been isolated from a methanolic extract of fresh leaves of Cestrum diurnum and the structure, (25 R)-5 alpha-spirostan-3beta-yl-4-O-[2-O-(beta-D-gluco pyranosyl)-beta-D-glucopyranosyl]-3-O-[beta-D-xylopyranosyl]-beta-D- glucopyranosyl]-beta-D-galactopyranoside, was determined by modern NMR technique and chemical reactions.

  3. Steroidal Saponins from Furcraea hexapetala Leaves and Their Phytotoxic Activity.


    Calle, Juan M; Pérez, Andy J; Simonet, Ana M; Guerra, José O; Macías, Francisco A


    Four new steroidal saponins (1-4) along with 13 known saponins were isolated from the leaves of Furcraea hexapetala. The new compounds were identified as (20R,22R,25R)-3β-hydroxy-5α-spirostan-12-one 3-O-{α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-O-[β-d-glucopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (1), (25R)-3β-hydroxy-5α-spirost-20(21)-en-12-one 3-O-{α-l-rhamnopyranosyl-(1→4)-O-β-d-glucopyranosyl-(1→3)-O-[β-d-glucopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (2), (25R)-5α-spirostan-3β-ol 3-O-{β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→2)-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (3), and (25R)-5β-spirostan-3β-ol 3-O-{β-d-glucopyranosyl-(1→6)-O-β-d-galactopyranoside} (4) by spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry, and chemical methods. The phytotoxicity of the isolated compounds against the standard target species Lactuca sativa was evaluated. Structure-activity relationships for these compounds with respect to phytotoxic effects are discussed.

  4. A new acylated anthocyanin from the red flowers of Camellia hongkongensis and characterization of anthocyanins in the section Camellia species.


    Li, Jian-Bin; Hashimoto, Fumio; Shimizu, Keiichi; Sakata, Yusuke


    Twelve anthocyanins (1-12) were isolated from the red flowers of Camellia hongkongensis Seem. by chromatography using open columns. Their structures were elucidated on the basis of spectroscopic analyses, that is, proton-nuclear magnetic resonance, carbon 13-nuclear magnetic resonance, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, high resolution electrospray ionization mass and ultraviolet visible spectroscopies. Out of these anthocyanins, a novel acylated anthocyanin, cyanidin 3-O-(6-O-(Z)-p-coumaroyl)-beta-galactopyranoside (6), two known acylated anthocyanins, cyanidin 3-O-(6-O-(E)-p-coumaroyl)-beta-galactopyranoside (7) and cyanidin 3-O-(6-O-(E)-caffeoyl)-beta-galactopyranoside (8), and three known delphinidin glycosides (10-12) were for the first time isolated from the genus Camellia. Furthermore, pigment components in C. japonica L., C. chekiangoleosa Hu and C. semiserrata Chi were studied. The results indicated that the distribution of anthocyanins was differed among these species. Delphinidin glycoside was only detected in the flowers of C. hongkongensis, which is a special and important species in the section Camellia. Based on the characterization of anthocyanins in the section Camellia species, there is a close relationship among these species, and C. hongkongensis might be an important parent for creating new cultivars with bluish flower color.

  5. Combined application of macroporous resin and high speed counter-current chromatography for preparative separation of three flavonoid triglycosides from the leaves of Actinidia valvata Dunn.


    Qu, Liping; Xin, Hailiang; Su, Yonghua; Zheng, Guoyin; Ling, Changquan


    In this paper, the combined techniques of macroporous resin column chromatography and high speed counter-current chromatography were applied for preparative separation of flavonoid triglycosides from the leaves of Actinidia valvata Dunn, a famous Chinese medicinal herb. Twelve kinds of macroporous resins were investigated by adsorption and desorption tests. HPD-300 resin showed the maximum effectiveness and thus was selected for the first cleaning-up, in which 20% ethanol was used to remove the undesired constituents and 60% ethanol to elute the targets. The crude extract was then purified by high speed counter-current chromatography with the solvent system composed of ethyl acetate-n-butanol-water (2:1:3 and 4:1:5, v/v). Three flavonoid triglycosides, namely, kaempferol 3-O-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside, kaempferol 3-O-α-L-rhamnopyranosyl-(1→3)-(4-O-acetyl-α-L-rhamnopyranosyl)-(1→6)-β-D-galactopyranoside and kaempferol 3-O-α-L-rhamnopyranosyl-(1→3)-(2,4-di-O-acetyl-α-L-rhamnopyranosyl)-(1→6)-β-D-galactopyranoside, were obtained. The purities of the separated compounds were all over 95% as determined by HPLC area normalization method. Their chemical structures were confirmed by UV, MS, NMR, and the standards.

  6. Three new flavonol glycosides from Suaeda maritima.


    Abd El-Latif, Rasha R; Mansour, Ragaa M A; Sharaf, Mohamed; Farag, Ahmad


    Three new flavonol glycosides isolated from the 70% methanol extract of Suaeda maritima (Chenopodiaceae) were characterized based on spectroscopic and chemical methods as quercetin 3-O-α-l-rhamnopyranosyl(1″' → 6″)-β-d-galactopyranoside-7-O-β-d-glucopyranosyl(1″″' → 2″″)-glucopyranoside, kaempferol 3-O-α-l-rhamnopyranosyl(1″' → 6″)-β-d-galactopyranoside-7-O-β-d-glucopyranosyl(1″″' → 2″″)-glucopyranoside, and kaempferol 3-O-α-l-rhamnopyranosyl(1″' → 6″)-β-d-galactopyranoside-7-O-(2″″'-O-trans-feruloyl)-β-d-glucopyranosyl-(1″″' → 2″″)-β-d-glucopyranoside. In addition, four known compounds, namely, quercetin and kaempferol, methyl cis, trans-ferulate, and methyl trans-ferulate were identified. The plant extract and these compounds showed cytotoxic activity against the human tumor cell lines MCF7, HCT116, and HEPG2.

  7. Phytotoxic steroidal saponins from Agave offoyana leaves.


    Pérez, Andy J; Simonet, Ana M; Calle, Juan M; Pecio, Łukasz; Guerra, José O; Stochmal, Anna; Macías, Francisco A


    A bioassay-guided fractionation of Agave offoyana leaves led to the isolation of five steroidal saponins (1-5) along with six known saponins (6-11). The compounds were identified as (25R)-spirost-5-en-2α,3β-diol-12-one 3-O-{α-l-rhamnopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (1), (25R)-spirost-5-en-3β-ol-12-one 3-O-{α-l-rhamnopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (2), (25R)-spirost-5-en-3β-ol-12-one 3-O-{β-d-xylopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (3), (25R)-26-O-β-d-glucopyranosylfurost-5-en-3β,22α,26-triol-12-one 3-O-{α-l-rhamnopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (4) and (25R)-26-O-β-d-glucopyranosylfurost-5-en-3β,22α,26-triol-12-one 3-O-{β-d-xylopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (5) by comprehensive spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry and chemical methods. The phytotoxicity of the isolated compounds on the standard target species Lactuca sativa was evaluated.

  8. Bioactive steroidal saponins from Agave offoyana flowers.


    Pérez, Andy J; Calle, Juan M; Simonet, Ana M; Guerra, José O; Stochmal, Anna; Macías, Francisco A


    Bioguided studies of flowers of Agave offoyana allowed the isolation of five steroidal saponins never described previously, Magueyosides A-E (1-5), along with six known steroidal saponins (6-11). The structures of compounds were determined as (25R)-spirost-5-en-2α,3β-diol-12-one 3-O-{β-d-xylopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (1), (25R)-spirost-5-en-2α,3β-diol-12-one 3-O-{β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (2), (25R)-spirost-5-en-2α,3β,12β-triol 3-O-{β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (3), (25R)-5α-spirostan-2α,3β-diol-12-one 3-O-{β-d-xylopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (4), and (25R)-5α-spirostan-2α,3β-diol-9(11)-en-12-one 3-O-{β-d-xylopyranosyl-(1→3)-O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-O-β-d-galactopyranoside} (5), by comprehensive spectroscopic analysis, including one- and two-dimensional NMR techniques, mass spectrometry and chemical methods. The bioactivities of the isolated compounds on the standard target species Lactuca sativa were evaluated. A dose-dependent phytotoxicity and low dose stimulation were observed.

  9. Alveolar macrophages and eicosanoids but not neutrophils, mediate bronchoconstriction induced by FMLP in the guinea-pig.

    PubMed Central

    Boukili, M. A.; Bureau, M. F.; Lellouch-Tubiana, A.; Lefort, J.; Simon, M. T.; Vargaftig, B. B.


    1. N-formyl-methionyl-leucyl-phenylalanine (FMLP), when administered by aerosol to guinea-pigs, induced a dose-dependent bronchoconstriction (BC) with no overt effect on platelet and leukocyte blood counts. Repeated administration of FMLP by aerosol was followed by desensitization. 2. Electron microscopy studies showed that administration of FMLP by aerosol is accompanied by alveolar macrophage activation, accumulation and aggregation in the alveolar lumens. Non-degranulated eosinophils were observed in the lungs and a few platelet micro-aggregates in the pulmonary microvasculature. 3. No significant accumulation of 131I-labelled albumin, 111In-labelled neutrophils or 111Inlabelled platelets was detected in the lungs after the administration of FMLP by aerosol, whereas the intravenous administration was accompanied by an increase of extravascular albumin and significant neutrophil sequestration in the lungs. 4. Aspirin administered intravenously or by aerosol reduced significantly the BC induced by an aerosol of FMLP. By contrast, intravenous indomethacin reduced only BC induced by the sub-maximal dose of FMLP as an aerosol whereas, when administered by inhalation, it inhibited BC induced by FMLP administered either intravenously or by aerosol at all the concentrations tested. 5. FMLP induced a dose-dependent contraction of the guinea-pig trachea, which was not inhibited by indomethacin. 6. The dual cyclo-oxygenase/lipoxygenase inhibitor compound BW755C suppressed the BC induced by an aerosol of FMLP at all the concentrations used, whereas the histamine H1-antagonist mepyramine was inactive. 7. Leukocyte depletion with vinblastine failed to reduce BC induced by intravenous or an aerosol of FMLP. 8. Our studies indicate that: (a) FMLP administered by aerosol induces dose-dependent BC followed by desensitization, indicating that local mechanisms account for BC; (b) BC induced by i.v. FMLP, but not by its inhalation, is accompanied by albumin extravasation and

  10. Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab' fragments.

    PubMed Central

    Casey, J. L.; King, D. J.; Chaplin, L. C.; Haines, A. M.; Pedley, R. B.; Mountain, A.; Yarranton, G. T.; Begent, R. H.


    The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The cross-linkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy beta-emitter. We have also compared the above antibody forms conjugated to both 131I and 90Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37 degrees C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 90Y was observed. Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 10(5) Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 131I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 131I and 90Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for 131I-labelled antibodies. Because of the faster clearance, it should

  11. Characterization of a double-sided silicon strip detector autoradiography system

    SciTech Connect

    Örbom, Anders Ahlstedt, Jonas; Östlund, Karl; Strand, Sven-Erik; Serén, Tom; Auterinen, Iiro; Kotiluoto, Petri; Hauge, Håvard; Olafsen, Tove; Wu, Anna M.; Dahlbom, Magnus


    Purpose: The most commonly used technology currently used for autoradiography is storage phosphor screens, which has many benefits such as a large field of view but lacks particle-counting detection of the time and energy of each detected radionuclide decay. A number of alternative designs, using either solid state or scintillator detectors, have been developed to address these issues. The aim of this study is to characterize the imaging performance of one such instrument, a double-sided silicon strip detector (DSSD) system for digital autoradiography. A novel aspect of this work is that the instrument, in contrast to previous prototype systems using the same detector type, provides the ability for user accessible imaging with higher throughput. Studies were performed to compare its spatial resolution to that of storage phosphor screens and test the implementation of multiradionuclide ex vivo imaging in a mouse preclinical animal study. Methods: Detector background counts were determined by measuring a nonradioactive sample slide for 52 h. Energy spectra and detection efficiency were measured for seven commonly used radionuclides under representative conditions for tissue imaging. System dead time was measured by imaging {sup 18}F samples of at least 5 kBq and studying the changes in count rate over time. A line source of {sup 58}Co was manufactured by irradiating a 10 μm nickel wire with fast neutrons in a research reactor. Samples of this wire were imaged in both the DSSD and storage phosphor screen systems and the full width at half maximum (FWHM) measured for the line profiles. Multiradionuclide imaging was employed in a two animal study to examine the intratumoral distribution of a {sup 125}I-labeled monoclonal antibody and a {sup 131}I-labeled engineered fragment (diabody) injected in the same mouse, both targeting carcinoembryonic antigen. Results: Detector background was 1.81 × 10{sup −6} counts per second per 50 × 50 μm pixel. Energy spectra and

  12. A pilot study of monoclonal antibody cG250 and low dose subcutaneous IL-2 in patients with advanced renal cell carcinoma.


    Davis, Ian D; Liu, Zhanqi; Saunders, Wayne; Lee, Fook-Thean; Spirkoska, Violeta; Hopkins, Wendie; Smyth, Fiona E; Chong, Geoffrey; Papenfuss, Anthony T; Chappell, Bridget; Poon, Aurora; Saunder, Timothy H; Hoffman, Eric W; Old, Lloyd J; Scott, Andrew M


    The chimeric monoclonal antibody cG250 recognizes the CAIX/MN antigen. cG250 induces antibody-dependent cellular cytotoxicity (ADCC) responses in vitro that can be enhanced by IL-2. We studied the effects of adding daily low-dose subcutaneous IL-2 to cG250 for treatment of clear cell renal cell carcinoma (RCC). The primary endpoints of the trial were toxicity and immunological effects (human anti-chimeric antibodies [HACA], ADCC, natural killer [NK] and lymphokine-activated killer cell [LAK] activity); secondary endpoints were cG250 biodistribution and pharmacokinetics (PK) and tumour response rates. Eligible patients had unresectable metastatic or locally advanced clear cell RCC with measurable or evaluable disease. Nine patients were treated with six doses of cG250 (10 mg/m(2)/week, first and fifth doses trace-labelled with (131)I), and 1.25 x 10(6) IU/m(2)/day IL-2 for six weeks. Treatment was generally well tolerated with no adverse events attributable to cG250. Two patients required a 50% dose reduction of IL-2 due to toxicity. No HACA was detected. (131)I-labeled cG250 showed excellent targeting of tumour deposits. (131)I cG250 PK: T(1/2)alpha 20.16 +/- 6.59 h, T(1/2)beta 126.21 +/- 34.04 h, CL 39.67 +/- 23.06 mL/h, Cmax 5.12 +/- 0.86 microg/mL, V(1) 3.88 +/- 1.05 L. IL-2 did not affect cG250 PK. A trend for increased percentage of circulating CD3-/CD16+CD56+ NK cells was observed. Some patients showed enhanced ADCC or LAK activity. No antitumour responses were observed. In conclusion, weekly cG250 with daily low-dose subcutaneous IL-2 is well tolerated. IL-2 does not influence cG250 biodistribution or increase HACA.

  13. How do in-vitro release profiles of nanosuspensions from Alzet® pumps correspond to the in-vivo situation? A case study on radiolabeled fenofibrate.


    Hill, A; Breyer, S; Geissler, S; Mier, W; Haberkorn, U; Weigandt, M; Mäder, K


    In research and development sufficiently high and constant plasma levels of drug candidates are often requested, but simple solutions of hydrophobic drugs delivered from the commonly used micro-osmotic pumps cannot meet these demands. Nanosuspensions released from implanted osmotic devices can be a strategy to overcome this challenge but little is known about their pharmacokinetic behavior after subcutaneous application. In the current study, four different nanosuspension formulations containing iodinated fenofibrate were prepared, physicochemically characterized and investigated concerning their in-vitro release kinetics from osmotic pumps. One nanosuspension of lower viscosity exhibited thereby an unexpectedly first order release kinetics, whereas the higher viscous counterpart was released in the expected zero-order manner. To assess the relation of the in-vitro release kinetics to the in-vivo fate of nanosuspensions, various [(131)I] iodinated fenofibrate formulations were subcutaneously applied to mice. The biodistribution was followed by means of γ-scintigraphy and γ-scintillation. Two different nanosuspensions released from osmotic pumps were compared to bolus injections of a nanosuspension and an organic drug solution. The distribution and elimination of the bolus injected drug solution were almost completed within 48h. In contrast, a long lasting (>1week) depot at the injection site was formed by the bolus injected nanosuspension. Ex vivo examination of the organs showed a sustained, but exponential decrease of the radiolabel concentration. More constant drug levels in the organs were achieved within the nanosuspensions released from osmotic pumps. The organ levels of [(131)I] labeled fenofibrate were found to be more constant in case of the pump with the higher viscous nanosuspension in contrast to the lower viscous counterpart. However, the very different release profiles of the lower and higher viscous nanosuspension observed in-vitro were not

  14. Immunohistochemical analysis of intratumoral heterogeneity of [131I]cG250 antibody uptake in primary renal cell carcinomas.

    PubMed Central

    Steffens, M. G.; Oosterwijk, E.; Zegwaart-Hagemeier, N. E.; van't Hof, M. A.; Debruyne, F. M.; Corstens, F. H.; Boerman, O. C.


    In previous studies, highly heterogeneous uptake of 131I-labelled chimeric monoclonal antibody G250 ([131I]cG250) in primary renal cell carcinomas has been observed (intratumoral differences > factor 100). In this study, we investigated a possible correlation between intratumoral antibody uptake and four immunohistochemically determined parameters: G250 antigen expression, blood vessel density, neovascularization and percentage of viable tumour cells. Whole tumour slices of four different tumours were cut into 1-cm3 cubes, and in each cube the [131I]cG250 uptake was determined. The correlation between [131I]cG250 uptake and each individual parameter was determined in a multiple regression analysis. Additionally, the data were reanalysed after introducing arbitrary cut-off values for each parameter. If a sample showed expression of a parameter above the introduced threshold value, this sample fulfilled one condition. Subsequently, the Pearson correlation coefficients were calculated from [131I]cG250 uptake and the number of fulfilled conditions (0-3). All tumour samples with high [131I]cG250 uptake [> 0.1% of the injected dose per gram (ID g(-1))] showed high antigen expression (> 50%). However, not all samples with high antigen expression displayed high uptake. A statistically significant correlation between [131I]cG250 uptake and antigen expression was found (beta = 0.44, 0.69 and 0.74) in three out of four tumours analysed. Of the other determined parameters, no consistent correlation with [131I]cG250 uptake was found; only the percentage of viable tumour cells correlated significantly in two out of four tumours (beta = 0.80 and 0.26). Calculation of the Pearson correlation coefficients showed a statistically significant correlation between [131I]cG250 uptake and an increased number of fulfilled conditions in all tumours, indicating that each of the individual parameters contribute to the uptake of [131I]cG250. These observations indicate that high antigen

  15. Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.


    Li, Xiu-Mei; Luo, Xue-Gang; Si, Chuan-Ling; Wang, Nan; Zhou, Hao; He, Jun-Fang; Zhang, Tong-Cun


    Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-β-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs.

  16. Efficient synthesis of tyrosol galactosides by the β-galactosidase from Enterobacter cloacae B5.


    Qi, Tingting; Gu, Guofeng; Xu, Li; Xiao, Min; Lu, Lili


    In this work, the β-galactosidase from Enterobacter cloacae B5 (BgaB5) exhibited excellent transglycosylation activity toward tyrosol (p-hydroxyphenethyl alcohol) when using lactose as the glycosyl donor, generating a series of tyrosol glycosides with potential pharmacological properties. The effects of substrate concentration, temperature, pH, and reaction time on the transglycosylation reaction catalyzed by the enzyme BgaB5 were studied in detail. Three tyrosol derivatives were produced in a total high yield of 50.0% when incubating the enzyme with 250 mM tyrosol and 1000 mM lactose (pH 7.5) at 50 °C for 5 min. These derivatives were subsequently purified by column chromatography and preparative thin-layer chromatography. MS analysis of the purified compounds suggested one monogalactoside (M r 300) and two digalactoside derivatives (M r 462). The following NMR analysis further identified them to be p-hydroxyphenethyl β-D-galactopyranoside, p-hydroxyphenethyl β-D- galactopyranosyl-(1 → 3')-β-D-galactopyranoside, and p-hydroxyphenethyl β-D- galactopyranosyl-(1 → 6')-β-D-galactopyranoside, respectively. The yield of the tyrosol monogalactoside which was known to possess potent bioactivities reached 39.4%, higher than other enzymatic yields reported so far. The two digalactosides, which were expected to have potential applications for novel drug screening and discovery, were artificially obtained with 10.6% yield for the first time.

  17. Two Novel Spirostene Glycosides from Selaginella chrysocaulos and their Chemotaxonomic Significance.


    Kunert, Olaf; Swamy, Rumalla Chidananda; Kumar, Bobbala Ravi; Rao, Achanta Venkata Narasimha Appa; Nandi, Owi Ivar; Schuehly, Wolfgang


    During an investigation of the Indian heterosporous fern Selaginella chrysocaulos, two novel C28 spirostene monosides, chrysocauloside A and B were identified. Chrysocauloside A (1β,3β-dihydroxy-20S,22R-spirost-5-ene-1-yl β-D-glucopyranoside) and chrysocauloside B (1β,3β-dihydroxy-20S,22R-spirost-5-ene-1-yl β-D-galactopyranoside) are O-glycosylated at C-1 and each bear a methyl group at C-24 and C-25. Spectroscopic (NMR) and MS data for the new compounds are given and the taxonomic significance of the occurrence of spirostenes in the group of Lycophyta is discussed.

  18. Preparation of amino alcohols condensed with carbohydrates: Evaluation of cytotoxicity and inhibitory effect on NO production.


    Corrêa, Taís A; Reis, Elaine F C; Alves, Lívia L; Alves, Caio C S; Castro, Sandra B R; Dias, Alyria T; Taveira, Aline F; Le Hyaric, Mireille; Couri, Mara R C; Ferreira, Ana P; De Almeida, Mauro V


    This work reports the preparation of several amino alcohols condensed with d-arabinose, d-glucose, and d-galactose derivatives. These compounds were evaluated in vitro for their cytotoxicity and ability to decrease nitric oxide production in J774A.1 cells. Arabinofuranoside derivatives 5a, 5b and 5c showed a significant inhibition of nitric oxide production (>80% at 5 μg/mL), while the galactopyranoside derivative 8d showed a notable nitric oxide inhibitory activity (126% at 0.5 μg/mL).

  19. Structural variation of glycolipids from Meiothermus taiwanensis ATCC BAA-400 under different growth temperatures.


    Yang, Yu-Liang; Yang, Feng-Ling; Huang, Zih-You; Tsai, Yu-Hsuan; Zou, Wei; Wu, Shih-Hsiung


    A major glycolipid, alpha-Galf(1-3)-alpha-Galp(1-6)-beta-GlcpNAcyl(1-2)-alpha-Glcp(1-1)-2-acylalkyldiol, is obtained from Meiothermus taiwanensis. This novel glycolipid is found only when the bacterium grows above 62 degrees C, which is significantly different from those from the same bacteria incubated at 55 degrees C. Terminal galactofuranoside and 1,2-alkyldiol lipids replaced galactopyranoside and glycerol lipids, respectively, under increased growth temperature. This variation is likely necessary for bacteria for keeping the stable outer membrane and surviving under extreme environments.

  20. Acylated flavonol tri- and tetraglycosides in the flavonoid metabolome of Cladrastis kentukea (Leguminosae).


    Kite, Geoffrey C; Rowe, Emily R; Lewis, Gwilym P; Veitch, Nigel C


    The foliar metabolome of Cladrastis kentukea (Leguminosae) contains a complex mixture of flavonoids including acylated derivatives of the 3-O-rhamnosyl(1→2)[rhamnosyl(1→6)]-galactosides of kaempferol and quercetin and their 7-O-rhamnosides, together with an array of non-acylated kaempferol and quercetin di-, tri- and tetraglycosides. Thirteen of the acylated flavonoids, 12 of which had not been reported previously, were characterised by spectroscopic and chemical methods. Eight of these were the four isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) and their 7-O-α-l-rhamnopyranosides, and three were isomers of quercetin 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E/Z-p-coumaroyl-β-d-galactopyranoside) - the remaining 4Z isomer was identified by LC-UV-MS analysis of a crude extract. The final two acylated flavonoids characterised by NMR were the 3E and 4E isomers of kaempferol 3-O-α-l-rhamnopyranosyl(1→2)[α-l-rhamnopyranosyl(1→6)]-(3/4-O-E-feruloyl-β-d-galactopyranoside)-7-O-α-l-rhamnopyranoside while the 3Z and 4Z isomers were again detected by LC-UV-MS. Using the observed fragmentation behaviour of the isolated compounds following a variety of MS experiments, a further 18 acylated flavonoids were given tentative structures by LC-MS analysis of a crude extract. Acylated flavonoids were absent from the flowers of C. kentukea, which contained an array of non-acylated kaempferol and quercetin glycosides. Immature fruits contained kaempferol 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside and its 7-O-α-rhamnopyranoside as the major flavonoids with acylated flavonoids, different from those in the leaves, only present as minor constituents. The presence of acylated flavonoids distinguishes the foliar flavonoid metabolome of C. kentukea from that of a closely related legume, Styphnolobium japonicum, which contains a similar

  1. Anti-inflammatory activity of a novel flavonol glycoside from the Bauhinia variegata Linn.


    Yadava, R N; Reddy, V Madhu Sudhan


    Bauhinia variegata Linn. (Leguminosae) is commonly known as 'Kachnar' in Hindi. It is distributed almost throught India. Its powdered bark is traditionally used for tonic, astrain, ulcers. It is also useful in skin diseases. The roots are used as antidote to snake poison. The present article deals with the isolation and structural elucidation of a novel flavonol glycoside 5,7,3',4'-tetrahydroxy-3-methoxy-7-O-alpha-L-rhamnopyranosyl(1-->3)-O-beta-galactopyranoside (1) from the roots of Bauhinia Variegata and its structure was identified by spectral analysis and chemical degradations. The novel compound (1) showed anti-inflammatory activity.

  2. Prostate Cancer Evaluation: Design, Synthesis and Evaluation of Novel Enzyme-Activated Proton MRI Contrast Agents

    DTIC Science & Technology


    commonly used reporter system.[34] However, the well-established chromogenic or fluorogenic substrates, relying on the hydrolysis by β-gal to release...M7, M9 and M10, respectively, in sodium phosphate buffer solution (PBS) (0.1 M, pH=7.4) in the presence of ferric ammonia citrate (FAC) with β...with yellow o- nitrophenyl β-D- galactopyranoside. (d) Western blot analysis: Protein was extracted from PC3 tumor cells and was quantified by a

  3. Flavonol galactoside caffeiate ester and homoisoflavones from Caesalpinia millettii HOOK. et ARN.


    Chen, Ping; Yang, Jun-Shan


    Chemical examination of the stems of Caesalpinia millettii HOOK. et ARN. led to the isolation of new flavonol glycoside caffeiate ester (1) and homoisoflavone (2), along with four known homoisoflavones: eucomin (3), bonducellin (4), 8-methoxybonducellin (5) and intricatinol (6). The structures of 1 and 2 were established to be tamarixetin 3-O-(6''-O-E-caffeoyl)-beta-D-galactopyranoside (1) and (Z)-7-hydroxy-8-methoxy-3-(4-methoxybenzyl) chroman-4-one (2) on the basis of detailed analyses of physical, chemical, and spectral data. Compounds 3-6 were isolated from this plant for the first time.

  4. Rapid biochemical screening for Salmonella, Shigella, Yersinia, and Aeromonas isolates from stool specimens.


    De Ryck, R; Struelens, M J; Serruys, E


    Four screens for the rapid (4 to 6 h) biochemical detection of pathogens from enteric isolation media are described. The Salmonella screen consisted of Kligler iron agar (KIA), motility-indole-urea-tryptophan-deamination semisolid medium (MIU-TDA), and the o-nitrophenyl-beta-D-galactopyranoside (ONPG) test; the Shigella screen consisted of KIA, MIU-TDA, the ONPG test, and the lysine decarboxylation-indole test; the Yersinia screen consisted of a rhamnose broth; the Aeromonas screen consisted of a xylose agar plate. When tested on 2,102 fresh isolates and 71 stock strains, the screens correctly detected 212 enteric pathogens (sensitivity, 100%), with a specificity of 98.1%.

  5. Three Dimensional Immobilization of Beta-Galactosidase on a Silicon Surface (Preprint)

    DTIC Science & Technology


    nm caused by the hydrolysis of o-nitrophenyl-β-D-galactopyranoside (oNPG). The reaction mixture contained 20 mM oNPG and 1 mM MgCl2 in potassium...incubated with 2 mL of a 1/2000 dilution of monoclonal anti-β- galactosidase antibody in phosphate buffered saline (PBS) for the detection of native or...Shu-gui, C. 1991. Development of biosensors for the simultaneous determination of sucrose and glucose, lactose and glucose, and starch and glucose

  6. Neisseria lactamica meningitis following skull trauma.


    Denning, D W; Gill, S S


    A woman developed meningitis due to Neisseria lactamica in association with a cribriform plate fracture. Cerebrospinal fluid antigen tests for Neisseria meningitidis were negative. The patient recovered with intravenous penicillin therapy. N. lactamica can be rapidly distinguished from N. meningitidis by the hydrolysis of ONPG (o-nitrophenyl-beta-D-galactopyranoside). In contrast to N. meningitidis and Neisseria gonorrhoeae, N. lactamica lacks virulence properties. As 100% of N. lactamica strains are susceptible to penicillin and all three previously described patients with N. lactamica meningitis have recovered with penicillin treatment, the reason for distinguishing the organisms in this context is primarily to prevent unnecessary anxiety and prophylaxis among contacts.

  7. Screening of Panamanian Plant Extracts for Pesticidal Properties and HPLC-Based Identification of Active Compounds

    PubMed Central

    Guldbrandsen, Niels; De Mieri, Maria; Gupta, Mahabir; Seiser, Tobias; Wiebe, Christine; Dickhaut, Joachim; Reingruber, Rüdiger; Sorgenfrei, Oliver; Hamburger, Matthias


    A library of 600 taxonomically diverse Panamanian plant extracts was screened for fungicidal, insecticidal, and herbicidal activities. A total of 19 active extracts were submitted to HPLC-based activity profiling, and extracts of Bocconia frutescens, Miconia affinis, Myrcia splendens, Combretum aff. laxum, and Erythroxylum macrophyllum were selected for the isolation of compounds. Chelerythrine (2), macarpine (3), dihydrosanguinarine (5), and arjunolic acid (8) showed moderate-to-good fungicidal activity. Myricetin-3-O-(6’’-O-galloyl)-β-galactopyranoside (13) showed moderate insecticidal activity, but no compound with herbicidal activity was identified. PMID:26839818

  8. A new steroidal saponin with antiinflammatory and antiulcerogenic properties from the bulbs of Allium ampeloprasum var. porrum.


    Adão, Camila Rodrigues; da Silva, Bernadete Pereira; Parente, José Paz


    A new steroidal saponin was isolated from the bulbs of Allium ampeloprasum var. porrumL. On the basis of chemical evidence, comprehensive spectroscopic analyses and comparison of known compounds, its structure was established as (3β,5α,6β,25R)-6-[(β-D-glucopyranosyl)oxy]-spirostan-3-yl O-β-D-glucopyranosyl-(1→2)-O-[β-D-glucopyranosyl-(1→3)]-β-D-galactopyranoside. Results of the present study indicated that the steroidal saponin showed haemolytic effects in the in vitro assays and demonstrated antiinflammatory activity and gastroprotective property using in vivo models.

  9. A new steroidal saponin from Agave attenuata.


    Mendes, Tatiana Paz; Silva, Graziela de Medeiros; da Silva, Bernadete Pereira; Parente, José Paz


    A new steroidal saponin was isolated from the leaves of Agave attenuata. Its structure was established as (3beta,beta,25S)-spirostan-3-yl O-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranosyl-(1 --> 2)-O-[beta-D-glucopyranosyl-(1 --> 3)]-beta-D-glucopyranosyl-(1 --> 4)-beta-D-galactopyranoside. The structural identification was performed using detailed analyses of 1H- and 13C-NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR, and COLOC) and chemical conversions. The hemolytic activity of the steroidal saponin was evaluated using an in vitro assay.

  10. Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.


    Salehi, Siamak; Eckley, Lorna; Sawyer, Greta J; Zhang, Xiaohong; Dong, Xuebin; Freund, Jean-Noel; Fabre, John W


    Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

  11. Photometric and electrochemical enzyme-multiplied assay techniques using beta-galactosidase as reporter enzyme.


    Ko, Francis H; Monbouquette, Harold G


    Beta-galactosidase (beta-gal) is shown to be a versatile new reporter enzyme in both photometric and electrochemical enzyme-multiplied assay techniques (EMATs). The well-known beta-gal substrate analog, o-nitrophenyl beta-d-galactopyranoside, yields the visibly colored, o-nitrophenol product upon hydrolysis, whereas the substrate, p-aminophenyl beta-D-galactopyranoside, gives rise to an electrooxidizable product, p-aminophenol. These beta-gal substrates made possible the demonstration of both photometric and electrochemical signal transduction schemes for beta-gal-based EMAT detection of estradiol (as the estradiol-bovine serum albumin (E-BSA) conjugate). The EMAT system is composed of the reporter enzyme, beta-gal, with covalently attached estradiol, and estrogen antibody, which inhibits enzyme activity of the beta-gal-estradiol conjugate up to approximately 75%. Reporter enzyme inhibition is relieved significantly by addition of < or =2 ng/mL of estradiol (as E-BSA), which competes for binding with the antibody. Thus, the presence of analyte (E-BSA) is reported by the enzyme (beta-gal), which amplifies the ligand-protein dissociation event by turning over its substrate repeatedly. The electrochemical version of EMAT, based on amperometric detection of p-aminophenol, is responsive to added estradiol within minutes. These results show that beta-gal may serve as a useful alternative to glucose-6-phosphate dehydrogenase, which currently is used as reporter enzyme in commercially available EMAT systems.

  12. Specific Detection of Antigen-Binding Cells by Localized Growth of Bacteria

    PubMed Central

    Rotman, Boris; Cox, David R.


    A new method for the enumeration of lymphoid cells with specific surface-receptors for antigen is described, based on the use of β-D-galactosidase (EC, either directly as an antigen or as a conjugated antigen. Binding of β-D-galactosidase is revealed by its activity in releasing riboflavin from a synthetic substrate, riboflavin-β-D-galactopyranoside. The riboflavin, inactive as a vitamin in the galactosidic form, becomes active when released by the enzyme, and can be detected by bioassay. Hence, lymphoid cells with receptors for β-D-galactosidase on their surface can be detected after they have been exposed to the enzyme, washed, and then plated in agar containing riboflavin-β-D-galactopyranoside, streptomycin, riboflavin-deficient medium, and a streptomycin-resistant strain of Streptococcus faecalis that requires riboflavin. Release of riboflavin is signalled by the growth of characteristic bacterial colonies over the cell that bound β-D-galactosidase. Images PMID:5002817

  13. Phytochemical Study of Tapirira guianensis Leaves Guided by Vasodilatory and Antioxidant Activities.


    Rodrigues, Amélia M G; Guimarães, Denise O; Konno, Tatiana U P; Tinoco, Luzineide W; Barth, Thiago; Aguiar, Fernando A; Lopes, Norberto P; Leal, Ivana C R; Raimundo, Juliana M; Muzitano, Michelle F


    The aim of this research was to perform a phytochemical study of the methanol leaves extract of T. guianensis (MET) guided by vasodilatory and antioxidant activities. The chemical profile of MET and the ethyl acetate fraction (EA fraction) was determined by HPLC-UV-MS and EA fraction guided fractionation by reverse-phase chromatography. The vasorelaxant effects of MET, fractions, sub-fractions and constituents were assessed on rat aorta pre-contracted with phenylephrine. Antioxidant activity was evaluated by using a DPPH assay. The results show that MET-induced vasodilation was dependent on NO/cGMP; and that the PI3K/Akt pathway seems to be the main route involved in eNOS activation. The EA fraction showed greater vasodilatory and antioxidant potency and was submitted to further fractionation. This allowed the isolation and characterization of quercetin, quercetin 3-O-(6″-O-galloyl)-β-d-galactopyranoside and 1,4,6-tri-O-galloyl-β-d-glucose. Also, galloyl-HHDP-hexoside and myricetin deoxyhexoside were identified by HPLC-UV-MS. These compounds are being described for the first time for T. guianensis. 1,4,6-tri-O-galloyl-β-d-glucose and quercetin 3-O-(6″-O-galloyl)-β-d-galactopyranoside showed no vasodilatory activity. Quercetin and myricetin glycoside seems to contribute to the MET activity, since they have been reported as vasodilatory flavonoids. MET-induced vasodilation could contribute to the hypotensive effect of T. guianensis previously reported.

  14. Flavonol Glycosides from Euphorbia microsciadia Bioss. with their Immunomodulatory Activities

    PubMed Central

    Ghanadian, Syed Mustafa; Ayatollahi, Abdul Majid; Afsharypour, Suleiman; Hareem, Sumaira; Abdalla, Omer Mohamed; Jules Kezetas Bankeu, Jean


    Four known flavonoids: quercetin 3-O-β-D-rutinoside (Q3Rut), myricetin 3-O-β-D-galactopyranoside (M3Gal), quercetin 3-O-β-D-galactopyranoside (Q3Gal) and quercetin 3-O-β-D-glucopyranoside (Q3Glc), for the first time were isolated from aerial parts of Euphorbia microsciadia. The chemical structure of them was elucidated on the basis of 1 and 2 D-NMR spectra and different spectroscopic techniques. The immunomodulatory activities of isolated compounds were compared using standard T-cell proliferation assay. These data showed that lymphocyte suppression activity of flavonoids (1-4) were comparatively lower than prednisolon as a standard drug. Immunosuppressive activity of flavonoids with hydroxyl groups at both 3′-and 4′-positions in their B-ring (Q3Gal) were more than those with 3′-,4′-and 5′-hydroxyl substitution (M3Gal). In these compounds, Q3Gal showed the most inhibitory activity, whereas M3Gal showed the least lymphocyte antiprolifeartive activity. PMID:24250520

  15. Trehalose Analogues: Latest Insights in Properties and Biocatalytic Production

    PubMed Central

    Walmagh, Maarten; Zhao, Renfei; Desmet, Tom


    Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (α-d-glucopyranosyl α-d-galactopyranoside) or galactotrehalose (α-d-galactopyranosyl α-d-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. “Greener” alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis. PMID:26084050

  16. Hydrolysis and transglycosylation activity of a thermostable recombinant beta-glycosidase from Sulfolobus acidocaldarius.


    Park, Ah-Reum; Kim, Hye-Jung; Lee, Jung-Kul; Oh, Deok-Kun


    We expressed a putative beta-galactosidase from Sulfolobus acidocaldarius in Escherichia coli and purified the recombinant enzyme using heat treatment and Hi-Trap ion-exchange chromatography. The resultant protein gave a single 57-kDa band by SDS-PAGE and had a specific activity of 58 U/mg. The native enzyme existed as a dimer with a molecular mass of 114 kDa by gel filtration. The maximum activity of this enzyme was observed at pH 5.5 and 90 degrees C. The half-lives of the enzyme at 70, 80, and 90 degrees C were 494, 60, and 0.2 h, respectively. The hydrolytic activity with p-nitrophenyl(pNP) substrates followed the order p-nitrophenyl-beta-D-fucopyranoside > pNP-beta-D-glucopyranoside > pNP-beta-D-galactopyranoside > pNP-beta-D-mannopyranoside > pNP-beta-D-xylopyranoside, but not toward aryl-alpha-glycosides or pNP-beta-L-arabinofuranoside. Thus, the enzyme was actually a beta-glycosidase. The beta-glycosidase exhibited transglycosylation activity with pNP-beta-D-galactopyranoside, pNP-beta-D-glucopyranoside, and pNP-beta-D-fucopyranoside in decreasing order of activity, in the reverse order of its hydrolytic activity. The hydrolytic activity was higher toward cellobiose than toward lactose, but the transglycosylation activity was lower with cellobiose than with lactose.

  17. Crystallization products of risedronate with carbohydrates and their substituted derivatives.


    Kos, Jiri; Pentakova, Monika; Oktabec, Zbynek; Krejcik, Lukas; Mandelova, Zuzana; Harokova, Pavla; Hruskova, Jana; Pekarek, Tomas; Dammer, Ondrej; Tkadlecova, Marcela; Havlicek, Jaroslav; Vinsova, Jarmila; Kral, Vladimir; Dohnal, Jiri; Jampílek, Josef


    The gastrointestinal absorption of bisphosphonates is in general only about 1%. To address this problem mixtures of risedronate monosodium salt with twelve varied sugar alcohols, furanoses, pyranoses and eight gluco-, manno- and galactopyranoside derivatives as counterions were designed in an effort to prepare co-crystals/new entities with improved intestinal absorption. Crystalline forms were generated by means of kinetically and/or thermodynamically controlled crystallization processes. One hundred and fifty-two prepared samples were screened by means of FT-NIR and FT-Raman spectroscopy. No co-crystal was prepared, but noteworthy results were obtained. A new solid phase of risedronate monosodium salt generated in the presence of phenyl-β-d-galactopyranoside under thermodynamically controlled crystallization conditions was found and also characterized using solid state NMR spectroscopy, X-ray powder diffraction and differential scanning calorimetry. This new polymorph was named as form P. Interactions between risedronate monosodium salt and both carbohydrates were confirmed by means of molecular dynamics simulation. In the present study the relationships between the chemical structures of the studied compounds required for crystalline form change are discussed.

  18. Antifungal Saponins from the Maya Medicinal Plant Cestrum schlechtendahlii G. Don (Solanaceae).


    Ta, Chieu Anh Kim; Guerrero-Analco, J Antonio; Roberts, Elizabeth; Liu, Rui; Mogg, Christopher D; Saleem, Ammar; Otárola-Rojas, Marco; Poveda, Luis; Sanchez-Vindas, Pablo; Cal, Victor; Caal, Federico; Subramaniam, Rajagopal; Smith, Myron L; Arnason, John T


    Bioassay-guided fractionation of the crude extract (80% EtOH) of the leaves of Cestrum schlechtendahlii, a plant used by Q'eqchi' Maya healers for treatment of athlete's foot, resulted in the isolation and identification of two spirostanol saponins (1 and 2). Structure elucidation by MS, 1D-NMR, and 2D-NMR spectroscopic methods identified them to be the known saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-α-L-rhamnopyranosyl-(1 → 2)-β-D-galactopyranoside (1) and new saponin (25R)-1β,2α-dihydroxy-5α-spirostan-3-β-yl-O-β-D-galactopyranoside (2). While 2 showed little or no antifungal activity at the highest concentration tested, 1 inhibited growth of Saccharomyces cerevisiae (minimum inhibitory concentration (MIC) of 15-25 μM), Candida albicans, Cryptococcus neoformans, and Fusarium graminearum (MIC of 132-198 μM).

  19. Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport

    SciTech Connect

    Herzlinger, D.; Carrasco, N.; Kaback, H.R.


    Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant ''uncoupled'' for beta-galactoside/H/sup +/ symport are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H/sup +/ electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H/sup +/ influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent K/sub m/. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.

  20. Flavonoid constituents in the leaves of Myrica rubra sieb. et zucc. with anti-inflammatory activity.


    Kim, Han Hyuk; Kim, Dong Hee; Kim, Manh Heun; Oh, Myoeng Hwan; Kim, So Ra; Park, Kwang Jun; Lee, Min Won


    The leaves of Myrica rubra sieb. et zucc. have been used in oriental traditional medicine for the treatment of burns, skin diseases, and as an antidiarrheal in China, Japan, and Korea. Activity guided isolation of the leaves of M. rubra has led to the isolation of five flavonoid: myricetin (1), myricitrin (2), myricetin 3-O-(2″-O-galloyl)-α-L-rhamnopyranoside (3), myricetin 3-O-(2″-O-galloyl)-β-D-galactopyranoside (4), and quercetin 3-O-(2″-O-galloyl)-β-D-galactopyranoside (5). All isolates were evaluated for their antioxidant potency against the superoxide anion (O2 (-)), and compounds 3-5 showed potent scavenging activities with 50 % inhibition concentration (IC50) values compared to the positive control, allopurinol. Compounds 1-5 were evaluated as inhibitors of various macrophage functions involved in the inflammatory process. These five compounds significantly and dose dependently inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO), pro-inflammatory cytokines, and the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-stimulated RAW 264.7 macrophages. Our results suggest that galloyl flavonol glycosides (3-5) isolated from M. rubra might be beneficial for the treatment of inflammation-related diseases.

  1. Novel flavonol glycosides from the aerial parts of lentil (Lens culinaris).


    Żuchowski, Jerzy; Pecio, Łukasz; Stochmal, Anna


    While the phytochemical composition of lentil (Lens culinaris) seeds is well described in scientific literature, there is very little available data about secondary metabolites from lentil leaves and stems. Our research reveals that the aerial parts of lentil are a rich source of flavonoids. Six kaempferol and twelve quercetin glycosides were isolated, their structures were elucidated using NMR spectroscopy and chemical methods. This group includes 16 compounds which have not been previously described in the scientific literature: quercetin 3-O-β-D-glucopyranosyl(1→2)-β-D-galactopyranoside-7-O-β-D-glucuropyranoside (1), kaempferol 3-O-β-D-glucopyranosyl(1→2)-β-D-galacto-pyranoside-7-O-β-D-glucuropyranoside (3), their derivatives 4-10,12-15,17,18 acylated with caffeic, p-coumaric, ferulic, or 3,4,5-trihydroxycinnamic acid and kaempferol 3-O-{[(6-O-E-p-coumaroyl)-β-D-glucopyranosyl(1→2)]-α-L-rhamnopyranosyl(1→6)}-β-D-galactopyranoside-7-O-α-L-rhamnopyranoside (11). Their DPPH scavenging activity was also evaluated. This is probably the first detailed description of flavonoids from the aerial parts of lentil.

  2. Microvascular effects of selective prostaglandin analogues in the eye with special reference to latanoprost and glaucoma treatment.


    Stjernschantz, J; Selén, G; Astin, M; Resul, B


    Prostaglandin F(2alpha) analogues have recently been introduced on the market for glaucoma treatment. While these drugs have a well-documented intraocular pressure reducing effect only a limited number of studies have been published regarding their effects on the microvasculature in the eye. Since many naturally occurring prostaglandins have marked effects on the cardiovascular system it is conceivable that synthetic prostaglandins used as glaucoma drugs may exert microvascular effects in the eye, even if they exhibit receptor selectivity. Latanoprost, the active principle of Xalatan((R)) eye drops, is a selective FP prostanoid receptor agonist, and much of the paper is focused on the microvascular effects of latanoprost and some closely related prostaglandin analogues. The purpose of the paper is to review the literature on the microvascular effects of prostaglandins in the eye, and to present some unpublished data on the effects of selective prostaglandin analogues. Most of the prostaglandin analogues studied exhibit selectivity for the FP prostanoid receptor. Results from studies with the following prostaglandin analogues are presented in the paper: PGF(2alpha)-isopropyl ester (PGF(2alpha)-IE), 17-phenyl-18,19,20-trinor-PGF(2alpha)-isopropyl ester (17-phenyl-PGF(2a)-IE), 15-keto-17-phenyl-18,19, 20-trinor-PGF(2alpha)-isopropyl ester (15-keto-17-phenyl-PGF(2a)-IE), 13,14-dihydro-17-phenyl-18,19,20-trinor-PGF(2alpha)-isopropy l ester (latanoprost), 13,14-dihydro-15R,S-17-phenyl-18,19, 20-trinor-PGF(2alpha)-isopropyl ester (PhXA34), 17-phenyl-18,19, 20-trinor-PGE(2)-isopropyl ester (17-phenyl-PGE(2)-IE), and 19R-hydroxy-PGE(2) (19R-OH-PGE(2)). The regional blood flow has been determined with radioactively labelled microspheres, the blood volume with (51)Cr labelled erythrocytes and the capillary permeability to albumin with (125)I and (131)I labelled albumin. PGF(2alpha)-IE has been shown to exert marked microvascular effects in the rabbit anterior segment

  3. Low molecular weight thyroglobulin leading to a goiter in a 12-year-old girl.


    Silva, J E; Santelices, R; Kishihara, M; Schneider, A


    We characterized the abnormal thyroglobulin (TG) in the thyroid and serum of a 12-yr-old girl with a large sporadic multinodular goiter first noted at age 4 yr. She developed normally and had no clinical evidence of hypothyroidism. However, her serum T4 was less than 1.0 microgram/dl, T3 was 125 ng/dl, and TSH was 155 microU/ml. Serum PBI was 9.7 micrograms/dl, and more than 90% was not extractable with butanol. The 24-h radioactive iodine uptake was 55%, not dischargeable by perchlorate. Hormone formation was tested by the administration of 131I before surgery. [131I]T4 and [131I]T3, but not 131I-labeled iodotyrosines, were present in the thyroidal venous blood. Hydrolysis of 10,000 X g supernatants from three randomly obtained samples of the goiter revealed 66-77% of the 131I as iodotyrosines, 2-4% as iodothyronines, and 10-12% as undigestable material; the MIT to DIT ratio ranged from 3.1-8.7, and the T4 to T3 ratio ranged from 2.3-8.3. The TG level was 2.5 mg/g in the goiter and 9.4 micrograms/ml in the serum. The RIA displacement curves for the goiter and serum TG levels were both identical to the curve produced by normal human TG. The iodine contents of goiter and serum TG were 0.49% and 0.47% (wt/wt), respectively. The T4 to T3 ratio was lower in the goiter (approximately 5) than in the serum iodoprotein (approximately 45), whereas the calculation of the T4 to T3 ratio in the thyroidal secretion was less than 1. The goiter and serum TG bound normally to Concanavalin A, indicating that they contained carbohydrate. When either serum- or goiter-soluble proteins were gel-filtered (Bio-Gel A-5m), TG immunoreactivity and stable iodine elution profiles were the same, suggesting that no significant amounts of other iodoproteins were present in the thyroid or circulation. Both serum and goiter TG elution volumes corresponded to mol wt of approximately 9 X 10(4). A sedimentation rate of 10-11 S was found for both goiter and serum TG. An abnormally low mol wt of 8

  4. Engineered antibodies take center stage.


    Huston, J S; George, A J


    implications across many disciplines. The growth in antibody engineering was highlighted by the attendance of some 600 participants at the meeting, doubling that of the 1999 meeting. Dramatic clinical acceptance of monoclonal antibodies during the past two years has fostered this growth, with sales in 2000 of 1.8 billion dollars and projections for 2001 of 3 billion dollars. However, economic measures cannot begin to convey the medical revolution that is being effected by these first humanized and chimerized monoclonal antibodies. At this juncture, the 10 monoclonal antibody therapeutics in clinical use are of murine origin, of which 3 are entirely murine (OKT3, Mylotarg, 90Y-labeled Bexxar), 4 have been chimerized (human constant domains replacing murine) (ReoPro, Rituxan and its 131I-labeled analogue (Zevalin), Simulect, Remicade) and 3 were chimerized and humanized (human residues being substituted for at least some mouse-specific framework residues in VH and VL) (Zenapax, Herceptin, Synagis). Fully humanized anti-CD52 (CAMPATH-1H) has also been approved by the FDA for the treatment of B-cell chronic lymphocytic leukemia and should become available in late 2001. Humanization was initially developed by Dr. Greg Winter at the MRC Laboratory of Molecular Biology (Cambridge, UK), who presented the meeting's keynote address, "Antibodies as a Paradigm for Molecular Evolution". His pioneering work in antibody phage display libraries has been reformulated into a daring approach to develop truly novel proteins with genetically paired structural elements. He described studies in combinatorial protein engineering with enormous implications for both industrial and therapeutic applications of macromolecules.

  5. Antioxidant and free radical scavenging activity of flavonol glycosides from different Aconitum species.


    Braca, Alessandra; Fico, Gelsomina; Morelli, Ivano; De Simone, Francesco; Tomè, Franca; De Tommasi, Nunziatina


    Bioassay-guided fractionation by 1,1-diphenyl-2-dipicrylhydrazyl (DPPH) radical scavenging test of polar extracts of some Italian Aconitum species (A. napellus subsp. tauricum, A. napellus subsp. neomontanum, A. paniculatum, A. vulparia) led to the isolation of 13 flavonol glycosides: quercetin 3-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->2)-beta-glucopyranoside-7-O-alpha-rhamnopyranoside (1), kaempferol 3-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->2)-beta-glucopyranoside-7-O-alpha-rhamnopyranoside (2), quercetin 3-O-(6-trans-p-coumaroyl)-beta-glucopyranosyl-(1-->2)-beta-glucopyranoside-7-O-alpha-rhamnopyranoside (3), kaempferol 3-O-(6-trans-p-coumaroyl)-beta-glucopyranosyl-(1-->2)-beta-glucopyranoside-7-O-alpha-rhamnopyranoside (4), quercetin 7-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->3)-alpha-rhamnopyranoside-3-O-beta-glucopyranoside (5), kaempferol 7-O-(6-trans-caffeoyl)-beta-glucopyranosyl-(1-->3)-alpha-rhamnopyranoside-3-O-beta-glucopyranoside (6), kaempferol 7-O-(6-trans-p-coumaroyl)-beta-glucopyranosyl-(1-->3)-alpha-rhamnopyranoside-3-O-beta-glucopyranoside (7), kaempferol 3-O-beta-(2"-acetyl)galactopyranoside (8), kaempferol 3-O-beta-(2"-acetyl)galactopyranoside-7-O-alpha-arabinopyranoside (9), quercetin 3-O-beta-(2"-acetyl)galactopyranoside-7-O-alpha-arabinopyranoside (10), quercetin 3,7-di-O-alpha-rhamnopyranoside (11), kaempferol 3,7-di-O-alpha-rhamnopyranoside (12) and quercetin 3-O-beta-glucopyranoside-7-O-alpha-rhamnopyranoside (13). Their antioxidant activity (AA) was determined by measuring free radical scavenging activity by DPPH test and the coupled oxidation of beta-carotene and linoleic acid assay. The results showed that 5 is the most active compound in the DPPH free-radical scavenging test (IC(50) 1.9 microM) while in the coupled oxidation of beta-carotene and linoleic acid assay compound 1 has the highest inhibitory ratio after 1h (58.9%). Some structure-activity relationships on the AA were obtained.

  6. Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium.


    Temuujin, Uyangaa; Chi, Won-Jae; Park, Jae-Sun; Chang, Yong-Keun; Song, Jae Yang; Hong, Soon-Kwang


    Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T(7) promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni(2+)-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His(6)-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The K(m) and V(max) of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.

  7. Polyoxypregnane glycosides from Caralluma retrospiciens.


    Halaweish, Fathi T; Huntimer, Eric; Khalil, Ashaf T


    Six related polyoxypregnane glycosides were isolated and characterised from Caralluma retrospiciens leaves. The compounds were identified as 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[3-O-methyl-6-deoxy-beta-D-allopyranosyl-(1 --> 4)-beta-D-cymaropyranosyl-(1 --> 4)-]-beta-D-cymaropyranoside], 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-allopyranosyl-(1 --> 4)-beta-D-cymaropyranosyl-(1 --> 4)-beta-D-cymaropyranoside], 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranoside], 12beta-benzoyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 6)-beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranoside], 12beta-benzoyloxy-11alpha-isovaleroyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranosyl-(1 --> 4)-3-O-methyl-6-deoxy-beta-D-galactopyranoside], and 12beta-benzoyloxy-11alpha-isovaleroyloxy-8beta,14beta-dihydroxypregn-20-one-3-O-[beta-D-glucopyranosyl (1 --> 4)-3-O-methyl-6-deoxy-beta-D-allopyranosyl-(1 --> 4)-beta-D-cymaropyranosyl-(1 --> 4)-beta-D-cymaropyranoside]. The structures were determined by detailed analysis of one- and two-dimensional NMR spectra as well as by chemical means. The compounds showed cytotoxic activities towards brine shrimp having IC50 values of 1.19 x 10(-4), 8.83 x 10(-5), 2.64 x 10(-4), 2.26 x 10(-4), 2.39 x 10(-4) and 1.70 x 10(-4) M, respectively. This is the first report of the isolation of these compounds from a natural source.

  8. Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas

    NASA Astrophysics Data System (ADS)

    Bakunina, Irina; Balabanova, Larissa; Golotin, Vasiliy; Slepchenko, Lyubov; Isakov, Vladimir; Rasskazov, Valeriy


    The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the E. coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by 1H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction.

  9. Two new steroidal saponins from dried fermented residues of leaf-juices of Agave sisalana forma Dong No. 1.


    Ding, Y; Tian, R H; Yang, C R; Chen, Y Y; Nohara, T


    In a previous paper, we reported the isolation and structure determination of three new steroidal saponins, dongnosides C (3), D (2) and E (1) from the dried fermented residues of leaf-juices of Agave sisalana forma Dong No. 1. In a continuing study on this plant, two additional new major steroidal saponins, named dongnosides B (4) and A (5), were obtained. Their structures were characterized respectively as tigogenin 3-O-alpha-L-rhamonpyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->2)- [beta-D- glucopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-beta-D-galactop yranoside and 3-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->2)-[beta- D- xylopyranosyl-(1-->3)-beta-D-glucopyranosyl-(1-->3)]-beta-D- glucopyranosyl-(1-->4)-beta-D-galactopyranoside on the basis of chemical and physicochemical evidence.

  10. Developing a Biosensor for Estrogens in Water Samples: Study ofthe Real-time Response of Live Cells of the Estrogen-sensitive YeastStrain RMY/ER-ERE using Fluorescence Microscopy

    SciTech Connect

    Wozei, E.; Hermanowicz, S.W.; Holman, H-Y.N.


    Using a fluorescein di-{beta}-d-galactopyranoside (FDG) substrate we show that in live cells of an estrogen-sensitive yeast strain RMY/ER-ERE with human estrogen receptor (ER{alpha}) gene and the lacZ gene which encodes {beta}-galactosidase, the uptake of 17{beta}-estradiol (E2) and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximal enzyme-catalyzed product formation evident after about 30 min of exposure to E2. This finding which agrees with the well-known rates of enzyme-catalyzed reactions could have implications for shortening the duration of environmental sample screening and monitoring regimes using yeast-based estrogen assays, and the development of biosensors for environmental estrogens to complement quantification methods.

  11. Developing a Biosensor for Estrogens in Water Samples: Study ofthe Real-time Response of Live Cells of the Estrogen-sensitive YeastStrain RMY/ER-ERE using Fluorescence Microscopy

    SciTech Connect

    Wozei, E.; Hermanowicz, S.W.; Holman, H-Y.N.


    Using a fluorescein di-{beta}-D-galactopyranoside (FDG) substrate we show that in live cells of an estrogen-sensitive yeast strain RMY/ER-ERE with human estrogen receptor (ER{alpha}) gene and the lacZ gene which encodes {beta}-galactosidase, the uptake of 17 {beta}-estradiol (E2) and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximal enzyme-catalyzed product formation evident after about 30 minutes of exposure to E2. This finding which agrees with the well-known rates of enzyme-catalyzed reactions could have implications for shortening the duration of environmental sample screening and monitoring regimes using yeast-based estrogen assays, and the development of biosensors for environmental estrogens to complement quantification methods.

  12. Differentiation of Rubidiated Methyl-d-Glycoside Stereoisomers by Infrared Multiple-Photon Dissociation Spectroscopy in the O-H and C-H Stretching Regions.


    Pearson, Wright L; Contreras, Cesar; Powell, David; Berden, Giel; Oomens, Jos; Bendiak, Brad; Eyler, John R


    Four isomeric sugar methylglycosides (α- and β-d-gluco- and galactopyranosides) were evaluated as rubidium cation coordination adducts in the gas phase using variable-wavelength multiple-photon dissociation in the range from 2750 to 3750 cm(-1). The adducts dissociated following photon absorption to yield neutral sugars and the rubidium cation, resulting in infrared "action" spectra. Well-resolved hydroxyl stretching bands clearly differentiate stereoisomers that vary solely in their asymmetry at single carbons. Density functional theory calculations of the lowest-energy gas-phase complexes indicate that rubidium coordinates with lone pairs of oxygen atoms as either bi- or tridentate complexes and that more than one positional coordination isomer could adequately account for most of the O-H stretch frequencies observed for each methylglycoside.

  13. Flavonoid glycosides from Prunus armeniaca and the antibacterial activity of a crude extract.


    Rashid, Fahima; Ahmed, Rehana; Mahmood, Azhar; Ahmad, Zaheer; Bibi, Nazia; Kazmi, Shahana Urooj


    Investigations on the chemical constituents of the fruits of Prunus armeniaca have led to the isolation of two new flavonoid glycosides, 4',5,7-trihydroxy flavone-7-O-[beta-D-mannopyranosyl (1'''-->2")]-beta-D-allopyranoside (1) and 3,4',5,7-tetrahydroxy-3',5'-di-methoxy flavone 3-O-[alpha-L-rhamnopyranosyl (1'''-->6")]-beta-D-galactopyranoside (2), from the butanolic fraction of the fruits. The butanolic extract exhibited antibacterial activity against both Gram positive and Gram negative bacteria. The structures of these compounds were elucidated through spectral studies, including 2D-NMR (COSY, NOESY, J-resolved), HMQC and HMBC experiments.

  14. Cloning and functional expression of a cDNA encoding coffee bean alpha-galactosidase.


    Zhu, A; Goldstein, J


    Purified coffee bean alpha-galactosidase (alpha Gal) has been used for removing terminal alpha-galactose residues from the glyco-conjugates at the red cell surface, in studies of blood group conversion. Here, we report the isolation and sequence of the full-length clone for coffee bean alpha Gal by using the polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) techniques. The cDNA clone (1.4 kb) contains a single open reading frame which encodes a protein of 378 amino acids (aa). Its authenticity is confirmed by perfect alignment of aa sequences obtained from purified coffee bean alpha Gal, and by immune reaction with the antibody raised against the enzyme. Furthermore, the protein produced in insect cells shows enzymatic activity towards a synthetic alpha Gal substrate, p-nitro-phenyl-alpha-galactopyranoside.

  15. Characterization of phenolic compounds in Brazilian pepper (Schinus terebinthifolius Raddi) exocarp.


    Feuereisen, Michelle M; Hoppe, Julia; Zimmermann, Benno F; Weber, Fabian; Schulze-Kaysers, Nadine; Schieber, Andreas


    The objective of this study was to characterize the phenolic composition of Brazilian pepper (Schinus terebinthifolius Raddi) exocarp extract. Using UHPLC-DAD-MS/MS analysis, four anthocyanins, three biflavonoids, gallic acid, and two types of hydrolyzable tannins (galloyl glucoses, galloyl shikimic acids) were tentatively identified. The structure of the so far unknown 7-O-methylpelargonidin 3-O-β-D-galactopyranoside was elucidated by 2D NMR. Within the group of gallotannins, galloyl shikimic acids with uncommon degrees of galloylation (tetra- to hexagalloyl shikimic acids) were detected. Among the biflavonoids, I3',II8-biapigenin (amentoflavone), I6,II8-biapigenin (agathisflavone), and II-2,3-dihydro-I3',II8-biapigenin were identified, which have already been described for Anacardiaceae. From the results of the present study together with previous findings on the phenolic profile of other Anacardiaceae plants, it is concluded that 7-methoxylated flavonoids are a chemotaxonomic trait frequently found in this family.

  16. Distinct chemotypes of Tephrosia vogelii and implications for their use in pest control and soil enrichment.


    Stevenson, Philip C; Kite, Geoffrey C; Lewis, Gwilym P; Forest, Félix; Nyirenda, Stephen P; Belmain, Steven R; Sileshi, Gudeta W; Veitch, Nigel C


    unrecorded flavonol glycosides present in T. candida were determined using cryoprobe NMR spectroscopy and MS as the 3-O-α-rhamnopyranosyl(1→6)-β-galactopyranoside-7-O-α-rhamnopyranoside, 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside, 3-O-α-rhamnopyranosyl(1→2)[α-rhamnopyranosyl(1→6)]-β-galactopyranoside-7-O-α-rhamnopyranoside, and 3-O-α-rhamnopyranosyl(1→2)[(3-O-E-feruloyl)-α-rhamnopyranosyl(1→6)]-β-galactopyranosides of 6-hydroxykaempferol 6-methyl ether. Tentative structures for a further 37 flavonol glycosides of T. candida were assigned by LC-MS/MS. The correct chemotype of T. vogelii (i.e. C1) needs to be promoted for use by farmers in pest control applications.

  17. Multigram-scale synthesis of an orthogonally protected 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT) building block.


    Christina, A E; Blas Ferrando, V M; de Bordes, F; Spruit, W A; Overkleeft, H S; Codée, J D C; van der Marel, G A


    Reported is the gram-scale synthesis of tert-butyldiphenylsilyl 4-(N-benzyloxycarbonyl)-amino-2-azido-2,4,6-trideoxy-β-D-galactopyranoside, which represents an orthogonally protected 2,4-diamino-D-fucose building block, a common constituent of various zwitterionic polysaccharides. The building block has been synthesized from D-glucosamine in 19% overall yield over 14 steps, requiring 5 chromatographic purifications. The key step in the synthesis is the introduction of the C-4 amino substituent, which has been accomplished by a one-pot three step procedure, involving regioselective C-3-O-trichloroacetimidate formation, C-4-O-triflation, and intramolecular substitution. The building block can be used as an acceptor and is readily transformed into a donor glycoside.

  18. Officinalioside, a new lignan glucoside from Borago officinalis L.


    Samy, Mamdouh Nabil; Hamed, Ashraf Nageeb El-Sayed; Sugimoto, Sachiko; Otsuka, Hideaki; Kamel, Mohamed Salah; Matsunami, Katsuyoshi


    A new lignan glucoside, officinalioside (1), was isolated from n-BuOH fraction of the aerial parts of Borago officinalis L., together with four known compounds: actinidioionoside (2), roseoside (3), crotalionoside C (4) and kaempferol 3-O-β-D-galactopyranoside (5). The structure of the new compound was established by means of spectroscopic and chemical analyses. Compounds 1 and 2 showed a moderate DPPH radical scavenging activity (IC50: 52.6 ± 1.70 and 41.3 ± 0.25 μM, respectively) comparable with that of the standard trolox (16.6 ± 2.2 μM) without any significant cytotoxicity towards human cell line A549 (IC50 > 100 μM).

  19. Antioxidant principles from the needles of red pine, Pinus densi fl ora.


    Jung, Mee Jung; Chung, Hae Young; Choi, Jin Ho; Choi, Jae Sue


    Antioxidant activity of Pinus densiflora Sieb. et Zucc. (Pinaceae) was evaluated for potential to inhibit hydroxyl radicals, inhibit total reactive oxygen species generation in kidney homogenates using 2',7'-dichlorodihydro fluorescein diacetate (DCHF-DA) and scavenge authentic peroxynitrites. The methanolic extract of P. densiflora showed strong antioxidant activity in the tested model systems and thus fractionated with several solvents. The antioxidant activity potential of the individual fraction was in the order of ethyl acetate > n-butanol > water > dichloromethane fraction. The ethyl acetate soluble fraction exhibiting strong antioxidant activity was further puri fi ed by repeated silica gel and Sephadex LH-20 column chromatographies. An active lignan (+)-isolarisiresinol xylopyranoside, as well as two active flavonoids [kaempferol 3-O-beta-galactopyranoside and its 6"-acetyl derivative], were isolated.

  20. [Glycosides from flowers of Jasminum officinale L. var. grandiflorum].


    Zhao, Gui-qin; Xia, Jing-jing; Dong, Jun-xing


    To study the chemical constituents of the flower of Jasminum officinale L. var. grandiflorum. The compounds were isolated and purified by re-crystallization and chromatography on silica gel and Sephadex LH-20 column. Their structures were elucidated on the physicochemical properties and spectral analysis. Seven glycosides were identified as kaempferol-3-O-alpha-L-rhamnopyranosyl (1-->3)-[alpha-L-rhamnopyranosyl (1-->6)]-beta-D-galactopyranoside (I), kaempferol-3-O-rutinoside (II), 7-ketologanin (III), oleoside-11-methyl ester (IV), 7-glucosyl-l1-methyl oleoside (V), ligstroside (VI), oleuropein (VII). Compound I is a new compound. Compounds III and V were isolated from the family of Jasminum for the first time and compounds II, IV and VI were isolated from Jasminum officinale L. var. grandiflorum for the first time.

  1. A new bioactive steroidal saponin from Agave attenuata.


    da Silva, Bernadete P; de Sousa, Allyne C; Silva, Graziela M; Mendes, Tatiana P; Parente, José P


    A new steroidal saponin was isolated from the leaves of Agave attenuata Salm-Dyck. Its structure was established as (3beta,5beta,22alpha,25S)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurostan-3-yl O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-O-[beta-D-glucopyranosyl-(1-->3)]-beta-D-glucopyranosyl-(1-->4)-beta-D-galactopyranoside. The structural identification was performed using detailed analyses of 1H and 13C NMR spectra including 2D NMR spectroscopic techniques (COSY, HETCOR and COLOC) and chemical conversions. The haemolytic potential of the steroidal saponin was evaluated and the anti-inflammatory activity was performed using the capillary permeability assay.

  2. Effect of the pH in the formation of β-galactosidase microparticles produced by a spray-drying process.


    Estevinho, Berta N; Ramos, Irena; Rocha, Fernando


    The objective of this work was to investigate the influence of pH in the microencapsulation process, using a modified chitosan to microencapsulate the enzyme β-galactosidase, by a spray-drying technique. Structural analysis of the surface of the particles was performed by scanning electron microscopy (SEM), showing that the obtained microparticles have an average diameter smaller than 3.5 μm and in general a regular shape. The activity of the enzyme was studied by spectrophotometric methods using the substrate O-nitrophenyl-β,D-galactopyranoside (ONPG). The parameters of Michaelis-Menten were calculated. The value of Km decreases with the decrease of the pH, which can be associated to an increase of the affinity between the enzyme and substrate to smaller pH's. The highest value of the parameter Vmax, representing the maximum reaction rate at a given enzyme concentration, was obtained at pH 6.

  3. Formation of Tryptophol Galactoside and an Unknown Tryptophol Ester in Euglena gracilis1

    PubMed Central

    Laćan, Goran; Magnus, Volker; Jeričević, Biserka; Kunst, Ljerka; Iskrić, Sonja


    The unicellular alga Euglena gracilis Klebs `Z' converted exogenous indole-3-ethanol (trytophol) to two major metabolites: tryptophol galactoside and an unknown compound, and to minor amounts of indole-3-acetic acid, tryptophol acetate, and tryptophol glucoside. The unknown was hydrolyzed to tryptophol by methanolic ammonia and should therefore be a tryptophol ester. The galactoside was identified as 2-(indol-3-yl)ethyl-β-d-galactopyranoside. This structure was established by comparison with an authentic standard involving chromatographic methods, ultraviolet and mass spectroscopy, enzymic and acid hydrolysis, and identification of the galactose in the hydrolysate. By forming tryptophol galactoside, Euglena differs from the higher plants examined so far, for which the corresponding glucoside is the only sugar conjugate of tryptophol detected. PMID:16663965

  4. New flavonol glycoside from Scabiosa prolifera L. aerial parts with in vitro antioxidant and cytotoxic activities.


    Al-Qudah, Mahmoud A; Otoom, Noor K; Al-Jaber, Hala I; Saleh, Ayman M; Abu Zarga, Musa H; Afifi, Fatma U; Abu Orabi, Sultan T


    Phytochemical investigation of the chemical constituents of the aerial parts of Scabiosa prolifera L. led to the isolation of one new flavonol glycoside, kaempferol-3-O-(4″,6″-di-E-p-coumaroyl)-β-D-galactopyranoside (1), along with ten other known compounds including luteolin-7-O-(2″-O-ethyl-β-glucopyranoside), β-sitosterol, β-sitosterylglucoside, ursolic acid, corosolic acid, ursolic acid 3-O-β-D-arabinopyranoside, apigenin, methyl-α-D-glucopyranoside, luteolin-7-O-β-glucopyranoside and isoorientin. The structures of all isolated compounds were established using chemical methods and spectroscopic methods including IR, UV, NMR (1D and 2D) and HRESIMS. All compounds were isolated for the first time from the plant. The antioxidant and cytotoxic activities of compounds 1 and 2 were also investigated.

  5. Metabolite profiling of the leaves of the Brazilian folk medicine Sideroxylon obtusifolium.


    Passos Oliveira, Adriana; Raith, Melanie; Kuster, Ricardo Machado; Rocha, Leandro Machado; Hamburger, Matthias; Potterat, Olivier


    Sideroxylon obtusifolium (Roem. & Schult.) T. D. Penn. (family Sapotaceae) is a tree native to Central and South America. Infusions of the bark and the leaves are used in Brazilian folk medicine as an anti-inflammatory remedy. However, information on the constituents of S. obtusifolium remains scarce, and only common pentacyclic triterpenoids have been previously reported. HPLC-DAD/MS analyses revealed that saponins and flavonoids were the main constituents of the leaves. From the butanol-soluble fraction of an ethanolic extract, a total of four saponins and ten flavonol glycosides were isolated by a combination of chromatographic methods including Sephadex LH-20, MPLC, and HPLC. Their structures were established by acid hydrolysis and spectroscopic methods, mainly MS (n), 1D and 2D NMR experiments. The compounds include the new triterpene glycoside 3-O-( β-D-glucopyranosyl)-protobassic acid 28-O- β-D-apiofuranosyl-(1 → 3)-O-[O- β-D-apiofuranosyl-(1 → 3)- β-D-xylopyranosyl-(1 → 4)]-O- α-L-rhamnopyranosyl-(1 → 2)- α-L-arabinopyranosyl ester ( 1), as well as the new flavonol glycosides, quercetin-3-O-(O- α-L-rhamnopyranosyl-(1→ 2)-O-[ β-D-glucopyranosyl-(1 → 3)]- β-D-galactopyranoside) ( 6) and kaempferol-3-O-(O- α-L-rhamnopyranosyl-(1 → 2)-O-[ β-D-glucopyranosyl-(1 → 3)]- β-D-galactopyranoside) ( 8). In addition, catechin and a glycerogalactolipid, gingerglycolipid A, were obtained from the ethyl acetate-soluble fraction. The isolated compounds could be used in the future as chemical markers for quality control of this herbal drug.

  6. Energetics of carbohydrate binding to Momordica charantia (bitter gourd) lectin: an isothermal titration calorimetric study.


    Sultan, Nabil Ali Mohammed; Swamy, Musti J


    Physico-chemical and carbohydrate binding studies have been carried out on the Momordica charantia (bitter gourd) seed lectin (MCL). The lectin activity is maximal in the pH range 7.4-11.0, but decreases steeply below pH 7.0. The lectin activity is mostly unaffected in the temperature range 4-50 degrees C, but a sharp decrease is seen between 50 and 60 degrees C, which could be correlated to changes in the structure of the protein as seen by circular dichroism and fluorescence spectroscopy. Isothermal titration calorimetric studies show that the tetrameric MCL binds two sugar molecules and the binding constants (Kb), determined at 288.15 K, for various saccharides were found to vary between 7.3 x 10(3) and 1.52 x 10(4)M(-1). The binding reactions for all the saccharides investigated were essentially enthalpy driven, with the binding enthalpies (DeltaHb) at 288.15 K being in the range of -50.99 and -43.39 kJ mol(-1), whereas the contribution to the binding reaction from the entropy of binding was negative, with values of binding entropy (DeltaSb) ranging between -99.2 and -72.0 J mol(-1)K(-1) at 288.15 K. Changes in heat capacity (DeltaCp) for the binding of disaccharides, lactose and lactulose, were significantly larger in magnitude than those obtained for the monosaccharides, methyl-beta-D-galactopyranoside, and methyl-alpha-D-galactopyranoside, and could be correlated reasonably well with the surface areas of these ligands. Enthalpy-entropy compensation was observed for all the sugars studied, suggesting that water structure plays an important role in the overall binding reaction. CD spectroscopy indicates that carbohydrate binding does not lead to significant changes in the secondary and tertiary structures of MCL, suggesting that the carbohydrate binding sites on this lectin are mostly preformed.

  7. Two distinct states of Escherichia coli cells that overexpress recombinant heterogeneous β-galactosidase.


    Zhao, Yun; He, Wei; Liu, Wei-Feng; Liu, Chun-Chun; Feng, Li-Kui; Sun, Lei; Yan, Yong-Bin; Hang, Hai-Ying


    The mechanism by which inclusion bodies form is still not well understood, partly because the dynamic processes of the inclusion body formation and its solubilization have hardly been investigated at an individual cell level, and so the important detailed information has not been acquired for the mechanism. In this study, we investigated the in vivo folding and aggregation of Aspergillus phoenicis β-D-galactosidase fused to a red fluorescence protein in individual Escherichia coli cells. The folding status and expression level of the recombinant β-D-galactosidase at an individual cell level was analyzed by flow cytometry in combination with transmission electron microscopy and Western blotting. We found that individual E. coli cells fell into two distinct states, one containing only inclusion bodies accompanied with low galactosidase activity and the other containing the recombinant soluble galactosidase accompanied with high galactosidase activity. The majority of the E. coli cells in the later state possessed no inclusion bodies. The two states of the cells were shifted to a cell state with high enzyme activity by culturing the cells in isopropyl 1-thio-β-D-galactopyranoside-free medium after an initial protein expression induction in isopropyl 1-thio-β-D-galactopyranoside-containing medium. This shift of the cell population status took place without the level change of the β-D-galactosidase protein in individual cells, indicating that the factor(s) besides the crowdedness of the recombinant protein play a major role in the cell state transition. These results shed new light on the mechanism of inclusion body formation and will facilitate the development of new strategies in improving recombinant protein quality.

  8. Flavonol pentaglycosides of Cordyla (Leguminosae: Papilionoideae: Swartzieae): distribution and taxonomic implications.


    Veitch, Nigel C; Kite, Geoffrey C; Lewis, Gwilym P


    A survey of foliar flavonoids in the swartzioid legume genus Cordyla s.l. revealed that three species, C. haraka, C. pinnata and C. richardii, were rich in flavonol pentaglycosides. Their structures were elucidated by spectroscopic and chemical methods as the 3-O-alpha-L-rhamnopyranosyl(1-->3)-alpha-L-rhamnopyranosyl(1-->2)[alpha-L-rhamnopyranosyl(1-->6)]-beta-D-galactopyranoside-7-O-alpha-L-rhamnopyranosides of quercetin and kaempferol (cordylasins A and B, respectively). These compounds were not found in the remaining species, C. africana, C. densiflora, C. madagascariensis (two subspecies) and C. somalensis, which exhibited different profiles of flavonoid glycosides. The distribution of flavonol pentaglycosides in Cordyla s.l. does not support a recent proposal to place both C. haraka and C. madagascariensis in the genus Dupuya [Kirkbride, J.H., 2005. Dupuya, a new genus of Malagasy legumes (Fabaceae). Novon 15, 305-314]. The generic relationship between Cordyla s.l. and Mildbraediodendron is also reassessed on the basis of chemical characters, as the O-linked tetrasaccharide that characterises cordylasins A and B is the same as that found in mildbraedin (kaempferol 3-O-alpha-l-rhamnopyranosyl(1-->3)-alpha-l-rhamnopyranosyl(1-->2)[alpha-l-rhamnopyranosyl(1-->6)]-beta-D-galactopyranoside), the main foliar flavonoid of Mildbraediodendron excelsum. Mildbraedin itself was found to be a minor constituent of leaflet extracts of C. haraka, C. pinnata and C. richardii, and a major constituent of C. somalensis.

  9. Terpenoids, flavonoids and caffeic acid derivatives from Salvia viridis L. cvar. Blue Jeans.


    Rungsimakan, Supattra; Rowan, Michael G


    Three diterpenoids, 1-oxomicrostegiol (1), viroxocin (2), viridoquinone (3), were isolated from the roots of Salvia viridis L. cvar. Blue Jeans. Five known diterpenoids, microstegiol (4), 7α-acetoxy-14-hydroxy-8,13-abietadiene-11,12-dione (5; 7-O-acetylhorminone tautomer), 7α,14-dihydroxy-8,13-abietadiene-11,12-dione (6; horminone tautomer), ferruginol and salvinolonyl 12-methyl ether (7) were also found in the roots together with 1-docosyl ferulate (8), and a mixture of 2-(4'-alkoxyphenyl) ethyl alkanoates (9). Two lupane triterpenoids, 2α-acetoxy-lup-20(29)-en-3β-ol (10), and 3β-acetoxy-lup-20(29)-en-2α-ol (11) were found in the aerial parts together with known compounds, lup-20(29)-ene-2α,3β-diol (12), ursolic acid, oleanolic acid, β-sitosterol and β-sitosterol glucoside. A known phenylpropanoid, trans-verbascoside (or acteoside; 13), was the main constituent in the polar fraction of the aerial part, and it is now reported in the genus Salvia for the first time. Other polyphenolic compounds were cis-verbascoside (14), leucosceptoside A (15), martynoside (16), caffeic acid, 6-O-caffeoyl-glucose (18), rosmarinic acid, salidroside, luteolin-7-O-α-rhamnopyranosyl-(1→6)-β-galactopyranoside, luteolin-7-O-β-galactopyranoside, luteolin-7-O-α-rhamnopyranosyl-(1→6)-β-glucopyranoside, luteolin-7-O-β-glucopyranoside, and apigenin-7-O-β-glucopyranoside. The structures were determined by 1D-, 2D-NMR and HR-ESI-MS techniques. Compounds 6, 10, ferruginol, ursolic acid and oleanolic acid exhibited antibacterial activity against Enterococcus faecalis (ATCC 775) with MIC 50 μM, 25 μM, 50 μM, 12.5 μM, 12.5 μM respectively. Ferruginol, ursolic acid and oleanolic acid were also active against Staphylococcus aureus (ATCC 6571), and Bacillus cereus (ATCC 2599) with MIC 12.5-50 μM. 4 was also active against S.aureus (ATCC 6571) with MIC 50 μM. These values are consistent with previous studies on the antimicrobial activity of Salvia diterpenoids.

  10. Detecting estrogenic activity in water samples withestrogen-sensitive yeast cells using spectrophotometry and fluorescencemicroscopy

    SciTech Connect

    Wozei, E.; Holman, H-Y.N.; Hermanowicz, S.W.; Borglin S.


    Environmental estrogens are environmental contaminants that can mimic the biological activities of the female hormone estrogen in the endocrine system, i.e. they act as endocrine disrupters. Several substances are reported to have estrogen-like activity or estrogenic activity. These include steroid hormones, synthetic estrogens (xenoestrogens), environmental pollutants and phytoestrogens (plant estrogens). Using the chromogenic substrate ortho-nitrophenyl-{beta}-D-galactopyranoside (ONPG) we show that an estrogen-sensitive yeast strain RMY/ER-ERE, with human estrogen receptor (hER{alpha}) gene and the lacZ gene which encodes the enzyme {beta}-galactosidase, is able to detect estrogenic activity in water samples over a wide range of spiked concentrations of the hormonal estrogen 17{beta}-estradiol (E2). Ortho-nitrophenol (ONP), the yellow product of this assay can be detected using spectrophotometry but requires cell lysis to release the enzyme and allow product formation. We improved this aspect in a fluorogenic assay by using fluorescein di-{beta}-D-galactopyranoside (FDG) as a substrate. The product was visualized using fluorescence microscopy without the need to kill, fix or lyse the cells. We show that in live yeast cells, the uptake of E2 and the subsequent production of {beta}-galactosidase enzyme occur quite rapidly, with maximum enzyme-catalyzed fluorescent product formation evident after about 30 minutes of exposure to E2. The fluorogenic assay was applied to a selection of estrogenic compounds and the Synchrotron-based Fourier transform infrared (SR-FTIR) spectra of the cells obtained to better understand the yeast whole cell response to the compounds. The fluorogenic assay is most sensitive to E2, but the SR-FTIR spectra suggest that the cells respond to all the estrogenic compounds tested even when no fluorescent response was detected. These findings are promising and may shorten the duration of environmental water screening and monitoring regimes using

  11. The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat.


    Garvie, E I; Cole, C B; Fuller, R; Hewitt, D


    Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).

  12. A method for determining beta-galactosidase activity of yogurt cultures in skim milk.


    Lin, W J; Savaiano, D A; Harlander, S K


    A method was developed for determining the specific activity of bacterial beta-galactosidase (EC during growth of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk. Individual and mixed strain cultures of S. thermophilus (St 3642, St14485) and L. bulgaricus (Lb11842, Lb880) were examined for growth (OD at 600 nm and viable cell counts), acid production, and beta-galactosidase activity (expressed as a function of recoverable TCA-precipitable cellular protein). Cultures were inoculated into 10% skim milk (2% inoculum) and incubated at 40 degrees C for 12 h. Aliquots were removed at 2-h intervals and diluted with ice cold EDTA, pH 12. The EDTA chelates calcium and solubilizes milk protein, allowing separation of the bacteria by centrifugation. Cells were then washed twice with 20 mM phosphate buffer and disrupted by sonication. Cell debris and intact cells were removed by centrifugation and the cell-free extract evaluated for beta-galactosidase activity using o-nitrophenyl-beta-D-galactopyranoside as substrate. Specific activities ranged from 0 to 6 units/mg protein. This simple and reproducible method is applicable for enzyme assays and measurement of cellular components where contamination by milk proteins is a potential problem.

  13. Induction and characterization of -galactosidase in an extreme thermophile.


    Ulrich, J T; McFeters, G A; Temple, K L


    A thermostable beta-galactosidase (EC; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.

  14. Use of a laboratory exercise on molar absorptivity to help students understand the authority of the primary literature.


    Soundararajan, Madhavan; Bailey, Cheryl P; Markwell, John


    To promote understanding of the authority of the primary literature in students taking our biochemistry laboratory courses, a biochemistry laboratory exercise on the determination of an acceptable molar absorptivity value of 2-nitrophenol (2-NP) was developed. This made the laboratory course much more relevant by linking to a thematic thread, β-galactosidase, that scaffolds concepts in one exercise with those in later exercises. The substrate for the continuous assay of β-galactosidase is the chromogenic 2-nitrophenyl-β-D-galactopyranoside that produces 2-NP. In an early laboratory exercise, students determine the wavelength of maximum absorption for the protonated and deprotonated form of 2-NP at various pH values and then determine the molar absorptivity of 2-NP. Students were encouraged to discuss apparent discrepancies not only in their own determinations of molar absorptivity values for 2-NP, but also in the published molar absorptivity values for 2-NP (2,150-21,300 M(-1) cm(-1) ) at almost the same pH and at 420 nm. Finally, the students were led to a publication that serves as an authentic source for molar absorptivity of 2-NP.

  15. Improving Properties of a Novel β-Galactosidase from Lactobacillus plantarum by Covalent Immobilization.


    Benavente, Rocio; Pessela, Benevides C; Curiel, Jose Antonio; de las Rivas, Blanca; Muñoz, Rosario; Guisán, Jose Manuel; Mancheño, Jose M; Cardelle-Cobas, Alejandra; Ruiz-Matute, Ana I; Corzo, Nieves


    A novel β-galactosidase from Lactobacillus plantarum (LPG) was over-expressed in E. coli and purified via a single chromatographic step by using lowly activated IMAC (immobilized metal for affinity chromatography) supports. The pure enzyme exhibited a high hydrolytic activity of 491 IU/mL towards o-nitrophenyl β-D-galactopyranoside. This value was conserved in the presence of different divalent cations and was quite resistant to the inhibition effects of different carbohydrates. The pure multimeric enzyme was stabilized by multipoint and multisubunit covalent attachment on glyoxyl-agarose. The glyoxyl-LPG immobilized preparation was over 20-fold more stable than the soluble enzyme or the one-point CNBr-LPG immobilized preparation at 50 °C. This β-galactosidase was successfully used in the hydrolysis of lactose and lactulose and formation of different oligosaccharides was detected. High production of galacto-oligosaccharides (35%) and oligosaccharides derived from lactulose (30%) was found and, for the first time, a new oligosaccharide derived from lactulose, tentatively identified as 3'-galactosyl lactulose, has been described.

  16. Human von Willebrand factor gene sequences target expression to a subpopulation of endothelial cells in transgenic mice.


    Aird, W C; Jahroudi, N; Weiler-Guettler, H; Rayburn, H B; Rosenberg, R D


    The present study was undertaken to define the 5' and 3' regulatory sequences of human von Willebrand factor gene that confer tissue-specific expression in vivo. Transgenic mice were generated bearing a chimeric construct that included 487 bp of 5' flanking sequence and the first exon fused in-frame to the Escherichia coli lacZ gene. In situ histochemical analyses in independent lines demonstrated that the von Willebrand factor promoter targeted expression of LacZ to a subpopulation of endothelial cells in the yolk sac and adult brain. LacZ activity was absent in the vascular beds of the spleen, lung, liver, kidney, testes, heart, and aorta, as well as in megakaryocytes. In contrast, in mice containing the lacZ gene targeted to the thrombomodulin locus, the 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside reaction product was detected throughout the vascular tree. These data highlight the existence of regional differences in endothelial cell gene regulation and suggest that the 733-bp von Willebrand factor promoter may be useful as a molecular marker to investigate endothelial cell diversity.

  17. Constituents from Polygonatum sibiricum and their inhibitions on the formation of advanced glycosylation end products.


    Wang, Juan; Lu, Cheng-Shu; Liu, Dong-Yan; Xu, Yan-Tong; Zhu, Yan; Wu, Hong-Hua


    A new saponin, isonarthogenin 3-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (1), together with twelve known compounds, were isolated from the rhizomes of Polygonatum sibiricum. The structures of these compounds were elucidated by analysis of their 1D/2D NMR and MS data. Among them, phenol compounds 4-7 and 9-10 showed significant inhibitions against the formation of advanced glycosylation end products, with IC50 values of 0.091 ± 0.0021, 0.10 ± 0.041, 0.014 ± 0.0027, 0.11 ± 0.011, 0.13 ± 0.045, and 0.055 ± 0.019 μM, respectively. The results will promote exploiting potential medicinal use of these compounds in the prevention of diabetic complications and supporting Polygonatum sibiricum as a functional food for healthy and medicinal diet.

  18. Glycosylation inhibition reduces cholesterol accumulation in NPC1 protein-deficient cells.


    Li, Jian; Deffieu, Maika S; Lee, Peter L; Saha, Piyali; Pfeffer, Suzanne R


    Lysosomes are lined with a glycocalyx that protects the limiting membrane from the action of degradative enzymes. We tested the hypothesis that Niemann-Pick type C 1 (NPC1) protein aids the transfer of low density lipoprotein-derived cholesterol across this glycocalyx. A prediction of this model is that cells will be less dependent upon NPC1 if their glycocalyx is decreased in density. Lysosome cholesterol content was significantly lower after treatment of NPC1-deficient human fibroblasts with benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside, an inhibitor of O-linked glycosylation. Direct biochemical measurement of cholesterol showed that lysosomes purified from NPC1-deficient fibroblasts contained at least 30% less cholesterol when O-linked glycosylation was blocked. As an independent means to modify protein glycosylation, we used Chinese hamster ovary ldl-D cells defective in UDP-Gal/UDP-GalNAc 4-epimerase in which N- and O-linked glycosylation can be controlled. CRISPR generated, NPC1-deficient ldl-D cells supplemented with galactose accumulated more cholesterol than those in which sugar addition was blocked. In the absence of galactose supplementation, NPC1-deficient ldl-D cells also transported more cholesterol from lysosomes to the endoplasmic reticulum, as monitored by an increase in cholesteryl [(14)C]-oleate levels. These experiments support a model in which NPC1 protein functions to transfer cholesterol past a lysosomal glycocalyx.

  19. The NMR studies on two new furostanol saponins from Agave sisalana leaves.


    Zou, Peng; Fu, Jing; Yu, He-shui; Zhang, Jie; Kang, Li-ping; Ma, Bai-ping; Yan, Xian-zhong


    The detailed NMR studies and full assignments of the 1H and 13C spectral data for two new furostanol saponins isolated from Agave sisalana leaves are described. Their structures were established using a combination of 1D and 2D NMR techniques including 1H, 13C, 1H-1H COSY, TOCSY, HSQC, HMBC and HSQC-TOCSY, and also FAB-MS spectrometry and chemical methods. The structures were established as (25S)-26-(beta-D-glucopyranosyl)-22 xi-hydroxyfurost-12-one-3beta-yl-O-alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)-O-[O-beta-D-glucopyranosyl-(1-->2)]-O-beta-D-glucopyranosyl-(1-->4)-beta-D-galacto- pyranoside (1) and (25S)-26-(beta-D-glucopyranosyl)-22xi-hydroxyfurost-5-en-12-one-3beta-yl-O-alpha-L-rhamno- pyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->3)-O-[O-beta-D-glucopyranosyl-(1-->2)]-O-beta-D-glucopyranosyl- (1-->4)-beta-D-galactopyranoside (2).

  20. Production of Lactase by Candida pseudotropicalis Grown in Whey.


    de Bales, S A; Castillo, F J


    Lactase (beta-d-galactosidase) was produced by Candida pseudotropicalis grown in deproteinized whey. Maximum enzyme production in 2% whey was obtained by supplementation with 0.15% yeast extract, 0.1% (NH(4))(2)SO(4), and 0.05% KH(2)PO(4) (wt/vol). Highest enzyme values (4.35 U/mg of cells and 68 U/ml) were obtained with 10 to 12% whey, while enzyme yield was maximal in 2% whey (0.87 U/mg of whey). Optimal initial pH for cultivation was 3.5. The best conditions for extraction included 2% (wt/vol) chloroform, 10 h of treatment, pH 6.6 and higher, and 30 to 37 degrees C. Optimum pH and temperature for enzyme activity were 6.2 and 47 degrees C. The enzyme had a K(m) for O-nitrophenyl-beta-d-galactopyranoside of 3.06 x 10 M and the initial V(max) was estimated as 6.63 x 10 M per min. It hydrolized 50 and 100% of the lactose in whey and milk within 4 and 5 h, respectively, at 37 degrees C. The lyophilized enzyme retained 95% of activity for 3 months when stored at -20 degrees C.

  1. β-galactosidase Production by Aspergillus niger ATCC 9142 Using Inexpensive Substrates in Solid-State Fermentation: Optimization by Orthogonal Arrays Design

    PubMed Central

    Kazemi, Samaneh; Khayati, Gholam; Faezi-Ghasemi, Mohammad


    Background: Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry, which is accomplished by enzyme β-galactosidase (β-gal, β-D-galactoside galactohydrolase, EC, trivial called lactase. Orthogonal arrays design is an appropriate option for the optimization of biotechnological processes for the production of microbial enzymes. Methods: Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was employed to screen the most significant levels of parameters, including the solid substrates (wheat straw, rice straw, and peanut pod), the carbon/nitrogen (C/N) ratios, the incubation time, and the inducer. The level of β-gal production was measured by a photometric enzyme activity assay using the artificial substrate ortho-Nitrophenyl-β-D-galactopyranoside. Results: The results showed that C/N ratio (0.2% [w/v], incubation time (144 hour), and solid substrate (wheat straw) were the best conditions determined by the design of experiments using the Taguchi approach. Conclusion: Our finding showed that the use of rice straw and peanut pod, as solid-state substrates, led to 2.041-folds increase in the production of the enzyme, as compared to rice straw. In addition, the presence of an inducer did not have any significant impact on the enzyme production levels. PMID:27721510

  2. Characterization of a salt-tolerant family 42 beta-galactosidase from a psychrophilic antarctic Planococcus isolate.


    Sheridan, P P; Brenchley, J E


    We isolated a gram-positive, halotolerant psychrophile from a hypersaline pond located on the McMurdo Ice Shelf in Antarctica. A phylogenetic analysis of the 16S rRNA gene sequence of this organism showed that it is a member of the genus Planococcus. This assignment is consistent with the morphology and physiological characteristics of the organism. A gene encoding a beta-galactosidase in this isolate was cloned in an Escherichia coli host. Sequence analysis of this gene placed it in glycosidase family 42 most closely related to an enzyme from Bacillus circulans. Even though an increasing number of family 42 glycosidase sequences are appearing in databases, little information about the biochemical features of these enzymes is available. Therefore, we purified and characterized this enzyme. The purified enzyme did not appear to have any metal requirement, had an optimum pH of 6.5 and an optimum temperature of activity at 42 degrees C, and was irreversibly inactivated within 10 min when it was incubated at 55 degrees C. The enzyme had an apparent K(m) of 4.9 micromol of o-nitrophenyl-beta-D-galactopyranoside, and the V(max) was 467 micromol of o-nitrophenol produced/min/mg of protein at 39 degrees C. Of special interest was the finding that the enzyme remained active at high salt concentrations, which makes it a possible reporter enzyme for halotolerant and halophilic organisms.

  3. A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria.


    Zhou, Junpei; Shi, Pengjun; Huang, Huoqing; Cao, Yanan; Meng, Kun; Yang, Peilong; Zhang, Rui; Chen, Xiaoyan; Yao, Bin


    The α-galactosidase gene, galA17, was cloned from Flavobacterium sp. TN17, a symbiotic bacterium isolated from the gut of Batocera horsfieldi larvae. The 2,205-bp full-length gene encodes a 734-residue polypeptide (GalA17) containing a putative 28-residue signal peptide and a catalytic domain belonging to glycosyl hydrolase family 36 (GH 36). The deduced amino acid sequence of galA17 was most similar to a putative α-galactosidase from Pedobacter sp. BAL39 (EDM38577; 66.6% identity) and a characterized α-galactosidase from Carnobacterium piscicola BA (AAL27305; 30.1%). Phylogenetic analysis revealed that GalA17 was similar to GH 36 α-galactosidases from symbiotic bacteria sharing two putative catalytic motifs, KWD and SDXXDXXXR, in which D480, S548, D549, and R556 were essential for α-galactosidase activity based on site-directed mutagenesis. Purified recombinant GalA17 showed apparent optimal activity at pH 5.5 and 45°C; exhibited strong resistance to digestion by trypsin, α-chymotrypsin, collagenase, and proteinase K; and efficiently hydrolyzed several synthetic and natural substrates (p-nitrophenyl-α-D-galactopyranoside, stachyose, melibiose, raffinose, soybean meal, locust bean gum, and guar gum).

  4. First Glycoside Hydrolase Family 2 Enzymes from Thermus antranikianii and Thermus brockianus with β-Glucosidase Activity

    PubMed Central

    Schröder, Carola; Blank, Saskia; Antranikian, Garabed


    Two glycoside hydrolase encoding genes (tagh2 and tbgh2) were identified from different Thermus species using functional screening. Based on amino acid similarities, the enzymes were predicted to belong to glycoside hydrolase (GH) family 2. Surprisingly, both enzymes (TaGH2 and TbGH2) showed twofold higher activities for the hydrolysis of nitrophenol-linked β-D-glucopyranoside than of -galactopyranoside. Specific activities of 3,966 U/mg for TaGH2 and 660 U/mg for TbGH2 were observed. In accordance, Km values for both enzymes were significantly lower when β-D-glucopyranoside was used as substrate. Furthermore, TaGH2 was able to hydrolyze cellobiose. TaGH2 and TbGH2 exhibited highest activity at 95 and 90°C at pH 6.5. Both enzymes were extremely thermostable and showed thermal activation up to 250% relative activity at temperatures of 50 and 60°C. Especially, TaGH2 displayed high tolerance toward numerous metal ions (Cu2+, Co2+, Zn2+), which are known as glycoside hydrolase inhibitors. In this study, the first thermoactive GH family 2 enzymes with β-glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to other enzymes of GH family 2. Our work contributes to a broader knowledge of substrate specificities in GH family 2. PMID:26090361

  5. Substrate-directed formation of small biocatalysts under prebiotic conditions.


    Kochavi, E; Bar-Nun, A; Fleminger, G


    One of the most debated issues concerning the origin of life, is how enzymes which are essential for existence of any living organism, evolved. It is clear that, regardless of the exact mechanism, the process should have been specific and reproducible, involving interactions between different molecules. We propose that substrate templating played a crucial role in maintaining reproducible and specific formation of prebiotic catalysts. This work demonstrates experimentally, for the first time, substrate-directed formation of an oligopeptide that possesses a specific catalytic activity toward the substrate on which it was formed. In our experiments we used the substrate O-nitrophenol-beta-D-galactopyranoside (ONPG) as a molecular template for the synthesis of a specific catalyst that is capable of cleaving the same substrate. This was achieved by incubation of the substrate with free amino acids and a condensing agent (dicyandiamide) at elevated temperatures. A linear increase with time of the reaction rate (d[product]/d2t), pointed to an acceleration regime, where the substrate generates the formation of the catalyst. The purified catalyst, produced by a substrate-directed mechanism, was analyzed, and identified as Cys2-Fe+2. The mechanism of substrate-directed formation of prebiotic catalysts provides a solution to both the specificity and the reproducibility requirements from any prebiotic system which should evolve into the biological world.

  6. Tracing cytotoxic effects of small organic Se species in human liver cells back to total cellular Se and Se metabolites.


    Marschall, T A; Kroepfl, N; Jensen, K B; Bornhorst, J; Meermann, B; Kuehnelt, D; Schwerdtle, T


    Small selenium (Se) species play a major role in the metabolism, excretion and dietary supply of the essential trace element selenium. Human cells provide a valuable tool for investigating currently unresolved issues on the cellular mechanisms of Se toxicity and metabolism. In this study, we developed two isotope dilution inductively coupled plasma tandem-mass spectrometry based methods and applied them to human hepatoma cells (HepG2) in order to quantitatively elucidate total cellular Se concentrations and cellular Se species transformations in relation to the cytotoxic effects of four small organic Se species. Species- and incubation time-dependent results were obtained: the two major urinary excretion metabolites trimethylselenonium (TMSe) and methyl-2-acetamido-2-deoxy-1-seleno-β-d-galactopyranoside (SeSugar 1) were taken up by the HepG2 cells in an unmodified manner and did not considerably contribute to the Se pool. In contrast, Se-methylselenocysteine (MeSeCys) and selenomethionine (SeMet) were taken up in higher amounts, they were largely incorporated by the cells (most likely into proteins) and metabolized to other small Se species. Two new metabolites of MeSeCys, namely γ-glutamyl-Se-methylselenocysteine and Se-methylselenoglutathione, were identified by means of HPLC-electrospray-ionization-Orbitrap-MS. They are certainly involved in the (de-)toxification modes of Se metabolism and require further investigation.

  7. Tryptophan environment, secondary structure and thermal unfolding of the galactose-specific seed lectin from Dolichos lablab: fluorescence and circular dichroism spectroscopic studies.


    Sultan, Nabil Ali Mohammed; Rao, Rameshwaram Nagender; Nadimpalli, Siva Kumar; Swamy, Musti J


    Fluorescence and circular dichroism spectroscopic studies were carried out on the galactose-specific lectin from Dolichos lablab seeds (DLL-II). The microenvironment of the tryptophan residues in the lectin under native and denaturing conditions were investigated by quenching of the intrinsic fluorescence of the protein by a neutral quencher (acrylamide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The results obtained indicate that the tryptophan residues of DLL-II are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains residing close to at least some of the tryptophan residues under the experimental conditions. Analysis of the far UV CD spectrum of DLL-II revealed that the secondary structure of the lectin consists of 57% alpha-helix, 21% beta-sheet, 7% beta-turns and 15% unordered structures. Carbohydrate binding did not significantly alter the secondary and tertiary structures of the lectin. Thermal unfolding of DLL-II, investigated by monitoring CD signals, showed a sharp transition around 75 degrees C both in the far UV region (205 nm) and the near UV region (289 nm), which shifted to ca. 77-78 degrees C in the presence of 0.1 M methyl-beta-D-galactopyranoside, indicating that ligand binding leads to a moderate stabilization of the lectin structure.

  8. Flavonol glycosides with lipid accumulation inhibitory activity and simultaneous quantitative analysis of 15 polyphenols and caffeine in the flower buds of Camellia sinensis from different regions by LCMS.


    Morikawa, Toshio; Ninomiya, Kiyofumi; Miyake, Sohachiro; Miki, Yoshinobu; Okamoto, Masaki; Yoshikawa, Masayuki; Muraoka, Osamu


    A simultaneous quantitative analytical method for 15 major polyphenols, e.g. five catechins (1-5) and 10 flavonols (6-15), as functional constituents in the extracts of "tea flowers", the flower buds of Camellia sinensis (Theaceae), has been developed. The content of caffeine (16), which showed similar chromatographic behaviour under the analytical conditions, was also determined. To approve the validity of the newly developed protocol, thirteen extracts of the plant's flower buds collected from different regions, i.e. China, Taiwan, Japan and India, were evaluated. The results indicated that the assay was reproducible and precise, and could be readily underutilised for the quality evaluation of tea flowers on the basis of polyphenols' contents. It was noteworthy that the contents of two major constituents, kaempferol 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside (10) and kaempferol 3-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→6)-β-D-galactopyranoside (11), varied by region where the flower buds were produced. A new flavonol glycoside, chakaflavonoside B (17), which was isolated in the course of this analytical study, was found to show oleic acid-albumin-induced lipid accumulation inhibitory activity.

  9. Enzyme kinetic measurements using a droplet-based microfluidic system with a concentration gradient.


    Bui, Minh-Phuong Ngoc; Li, Cheng Ai; Han, Kwi Nam; Choo, Jaebum; Lee, Eun Kyu; Seong, Gi Hun


    In this paper, we propose a microfluidic device that is capable of generating a concentration gradient followed by parallel droplet formation within channels with a simple T-junction geometry. Linear concentration gradient profiles can be obtained based on fluid diffusion under laminar flow. Optimized conditions for generating a linear concentration gradient and parallel droplet formation were investigated using fluorescent dye. The concentration gradient profile under diffusive mixing was dominated by the flow rate at sample inlets, while parallel droplet formation was affected by the channel geometry at both the inlet and outlet. The microfluidic device was experimentally characterized using optimal layout and operating conditions selected through a design process. Furthermore, in situ enzyme kinetic measurements of the β-galactosidase-catalyzed hydrolysis of resorufin-β-d-galactopyranoside were performed to demonstrate the application potential of our simple, time-effective, and low sample volume microfluidic device. We expect that, in addition to enzyme kinetics, drug screening and clinical diagnostic tests can be rapidly and accurately performed using this droplet-based microfluidic system.

  10. Use of a Modified α-N-Acetylgalactosaminidase in the Development of Enzyme Replacement Therapy for Fabry Disease

    PubMed Central

    Tajima, Youichi; Kawashima, Ikuo; Tsukimura, Takahiro; Sugawara, Kanako; Kuroda, Mayuko; Suzuki, Toshihiro; Togawa, Tadayasu; Chiba, Yasunori; Jigami, Yoshifumi; Ohno, Kazuki; Fukushige, Tomoko; Kanekura, Takuro; Itoh, Kohji; Ohashi, Toya; Sakuraba, Hitoshi


    A modified α-N-acetylgalactosaminidase (NAGA) with α-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-α-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease. PMID:19853240

  11. Potentiometric enzyme immunoassay using miniaturized anion-selective electrodes for detection

    PubMed Central

    Szűcs, Júlia; Pretsch, Ernö; Gyurcsányi, Róbert E.


    An enzyme-linked immunosorbent assay (ELISA) for prostate specific antigen (PSA) detection in human serum was developed based on the potentiometric detection of 6,8-difluoro-4-methylumbelliferone (DiFMU). The assays were carried out in anti-human PSA capture-antibody modified microtiter plates (150 µl volume). After incubation in the PSA containing serum samples, β-galactosidase-labeled PSA tracer antibody was added. The β-galactosidase label catalyzed the hydrolysis of 6,8-difluoro-4-methylumbelliferyl-β-D-galactopyranoside (DiFMUG) and the resulting DiFMU− anion was detected by potentiometric microelectrodes with anion-exchanger membrane. The selectivity of the anion-exchanger electrode is governed by the lipophilicity of the anions in the sample. Since DiFMU− is much more lipophilic (log P = 1.83) than any of the inorganic anions normally present in the working buffers and occurs in its anionic form at the physiological pH (pKa = 4.19), it was chosen as the species to be detected. The potentiometric ELISA-based method detects PSA in serum with a linear concentration range of 0.1–50 ng/mL. These results confirm the applicability of potentiometric detection in diagnostic PSA assays. Owing to simple methodology and low cost, potentiometric immunoassays seem to offer a feasible alternative to the development of in vitro diagnostic platforms. PMID:20448926

  12. A galactose-functionalized dendritic siRNA-nanovector to potentiate hepatitis C inhibition in liver cells.


    Lakshminarayanan, Abirami; Reddy, B Uma; Raghav, Nallani; Ravi, Vijay Kumar; Kumar, Anuj; Maiti, Prabal K; Sood, A K; Jayaraman, N; Das, Saumitra


    A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse 'off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated region (UTR) of HCV RNA using a liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand (DG). Physico-chemical characterization revealed finer details of complexation of DG with siRNA, whereas molecular dynamic simulations demonstrated sugar moieties projecting "out" in the complex. Preferential delivery of siRNA to the liver was achieved through a highly specific ligand-receptor interaction between dendritic galactose and the asialoglycoprotein receptor. The siRNA-DG complex exhibited perinuclear localization in liver cells and co-localization with viral proteins. The histopathological studies showed the systemic tolerance and biocompatibility of DG. Further, whole body imaging and immunohistochemistry studies confirmed the preferential delivery of the nucleic acid to mice liver. Significant decrease in HCV RNA levels (up to 75%) was achieved in HCV subgenomic replicon and full length HCV-JFH1 infectious cell culture systems. The multidisciplinary approach provides the 'proof of concept' for restricted delivery of therapeutic siRNAs using a target oriented dendritic nano-vector.

  13. Specific and reversible immobilization of proteins tagged to the affinity polypeptide C-LytA on functionalized graphite electrodes.


    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M; Climent, Víctor; Sanz, Jesús M


    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.

  14. Specific and Reversible Immobilization of Proteins Tagged to the Affinity Polypeptide C-LytA on Functionalized Graphite Electrodes

    PubMed Central

    Bello-Gil, Daniel; Maestro, Beatriz; Fonseca, Jennifer; Feliu, Juan M.; Climent, Víctor; Sanz, Jesús M.


    We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field. PMID:24498237

  15. A simple, robust enzymatic-based high-throughput screening method for antimicrobial peptides discovery against Escherichia coli.


    Thirumalai, Muthukumaresan Kuppuswamy; Roy, Arpita; Sanikommu, Suma; Arockiaraj, Jesu; Pasupuleti, Mukesh


    The indiscriminate usage of antibiotics has created a major problem in the form of antibiotic resistance. Even though new antimicrobial drug discovery programs have been in place from the last two decades, still we are unsuccessful in identifying novel molecules that have a potential to become new therapeutic agents for the treatment of microbial infections. A major problem in most screening studies is the requirement of high-throughput techniques. Given this, we present here an enzyme-based robust method for screening antimicrobial agent's active against Escherichia coli. This method is based upon the ability of the intracellular innate enzyme to cleave o-nitrophenyl β-d-galactopyranoside (non-chromogenic) to o-nitrophenolate (ONP) (chromogenic) upon the membrane damage or disruption. In comparison with the other currently available methods, we believe that our method provides an opportunity for real-time monitoring of the antimicrobial agents action by measuring the ONP generation in a user-friendly manner. Even though this method can be applied to other strain, our experience shows that one has to be careful especially when the pigments or metabolites present in the bacteria have the same wavelength absorbance.

  16. Microbead-based electrochemical immunoassay with interdigitated array electrodes.


    Thomas, Jennifer H; Kim, Sang Kyung; Hesketh, Peter J; Halsall, H Brian; Heineman, William R


    The objective of this study was to develop a sensitive and miniaturized immunoassay by coupling a microbead-based immunoassay with an interdigitated array (IDA) electrode. An IDA electrode amplifies the signal by recycling an electrochemically redox-reversible molecule. The microfabricated platinum electrodes had 25 pairs of electrodes with 1.6-microm gaps and 2.4-microm widths. An enzyme-labeled sandwich immunoassay on paramagnetic microbeads with mouse IgG as the analyte and beta-galactosidase as the enzyme label was used as the model system. beta-Galactosidase converted p-aminophenyl beta-D-galactopyranoside to p-aminophenol (PAP). This enzyme reaction was measured continuously by positioning the microbeads near the electrode surface with a magnet. Electrochemical recycling occurred with PAP oxidation to p-quinone imine (PQI) at +290 mV followed by PQI reduction to PAP at -300 mV vs Ag/AgCl. Dual-electrode detection amplified the signal fourfold compared to single-electrode detection, and the recycling efficiency reached 87%. A calibration curve of PAP concentration vs anodic current was linear between 10(-4) and 10(-6)M. A signal from 1000 beads in a 20-microL drop was detectable and the immunoassay was complete within 10 min with a detection limit of 3.5x10(-15)mol mouse IgG.

  17. Detection of hormone active chemicals using genetically engineered yeast cells and microfluidic devices with interdigitated array electrodes.


    Ino, Kosuke; Kitagawa, Yusuke; Watanabe, Tsuyoshi; Shiku, Hitoshi; Koide, Masahiro; Itayama, Tomoaki; Yasukawa, Tomoyuki; Matsue, Tomokazu


    Endocrine disruptors that act like hormones in the endocrine system might have toxic effects. Therefore, it is important to develop a portable device that can detect hormone active chemicals in samples rapidly and easily. In this study, a microfluidic device was developed for the detection of hormone active chemicals using genetically engineered yeast cells. The yeast cells were used as biosensors since they were genetically engineered to respond to the presence of hormone active chemicals by synthesizing beta-galactosidase (beta-gal). For achieving further sensitivity, we incorporated interdigitated array (IDA) electrodes (width, 1.2 microm; gap, 0.8 microm) with 40 electrode fingers into the analytical chamber of the microfluidic device. The yeast cells precultured with a hormone active chemical, 17beta-estradiol (E2), were trapped from the main channel of the device to the analytical camber by electrophoresis. After trapping in the analytical chamber, we performed electrochemical detection of beta-gal induced in the yeast cells with the IDA electrodes. Actually, electrochemical detection was performed on p-aminophenol that was converted from p-aminophenyl-beta-D-galactopyranoside with beta-gal. The electrochemical signals from the yeast cells precultured with 17beta-estradiol were successfully detected with the device. Furthermore, the inhibitory effects of antagonists such as tamoxifen were also detected electrochemically by using the device. Thus, the present microfluidic device can be used for highly sensitive detection of hormone active chemicals.

  18. Intracellular target for alpha-terthienyl photosensitization: involvement of lysosomal membrane damage.


    Sasaki, M; Koyama, S; Tokiwa, K; Fujita, H


    Intracellular targets for the photosensitizer alpha-terthienyl (alpha T) were examined by fluorescence microscopy and microfluorospectrometry using human nonkeratinized buccal cells. Intracellular distribution of alpha T was observed as fluorescent patches widely dispersed in the cytoplasm. The distribution of the fluorescent patches was compared with that of acid phosphatase activity visualized as an azo dye produced by the fast garnet 2-methyl-4-[(2-methyl-phenyl)azo]benzenediasonium sulfate reaction. Because both the distribution sites coincided, lysosomes were the likely sites of intracellular affinity of alpha T. However, because acid phosphatase is not a specific lysosomal marker, we tried to detect another lysosomal enzyme, beta-galactosidase, to confirm if the fluorescent patches were lysosomes, using fluorescein-di-(beta-D-galactopyranoside) (FDG) as a fluorogenic substrate. Without UV-A (320-400 nm) irradiation of the cells after uptake of alpha T and FDG, no significant fluorescence was observed. In contrast, with prior UV-A irradiation in the presence of alpha T and FDG, the bright yellow fluorescence of fluorescein, which is the digested product of FDG, was clearly detected in the cells by fluorescence microscopy. This observation implied that inflow of external FDG into the lysosomes is caused by lysosomal membrane damage on alpha T photosensitization. The present results indicated that lysosomes are the primary photosensitization site of alpha T.

  19. Characterization of lactose utilization and β-galactosidase in Lactobacillus brevis KB290, the hetero-fermentative lactic acid bacterium.


    Honda, Hiroyuki; Yajima, Nobuhiro; Saito, Tadao


    Unlike dairy lactic acid bacteria, Lactobacillus brevis cannot ferment milk. We characterized the lactose utilization by L. brevis KB290. In a carbohydrate fermentation assay using API 50 CHL, we showed during 7 days L. brevis did not ferment lactose. L. brevis grew to the stationary phase in 2 weeks in MRS broth containing lactose as the carbon source. L. brevis slowly consumed the lactose in the medium. L. brevis hydrolyzed lactose and a lactose analog, o-nitrophenyl-β-D-galactopyranoside (ONPGal). This β-galactosidase activity for ONPGal was not repressed by glucose, galactose, fructose, xylose, or maltose showing the microorganism may not have carbon catabolite repression. We purified the L. brevis β-galactosidase using ammonium sulfate precipitation and several chromatographies. The enzyme's molecular weight is estimated at 72 and 37 kDa using SDS-PAGE analysis. The N-terminal amino acid sequence of the larger protein was 90 % similar to the sequence of the putative β-galactosidase (YP_796339) and the smaller protein was identical to the sequence of the putative β-galactosidase (YP_796338) in L. brevis ATCC367. This suggests the enzyme is a heterodimeric β-galactosidase. The specific activity of the purified enzyme for lactose is 55 U/mg. We speculate inhibition of lactose transport delays the lactose utilization in L. brevis KB290.

  20. Isolation and characterization of guinea-pig serum amyloid P component.

    PubMed Central

    Maudsley, S; Hind, C R; Munn, E A; Buttress, N; Pepys, M B


    A pentraxin was isolated from acute-phase guinea-pig serum by calcium-dependent affinity chromatography on agarose. It was immunochemically identical to guinea-pig amyloid P component and therefore has been called guinea-pig serum amyloid P component (SAP). Guinea-pig SAP has an apparent MW of between 265,000 and 300,000 by different techniques, and is composed of 10 noncovalently associated subunits arranged in two pentameric annular discs interacting face-to-face. It is apparently composed of two types of subunit, which run as a closely spaced doublet on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). At least one type of subunit is glycosylated. The serum concentration was 16 +/- 4 mg/l in outbred animals, rising to 25 +/- 4 mg/l in an acute-phase response. Binding to agarose correlated with the agarose pyruvate content and was completely abolished by diazomethane treatment of the agarose, which methylates the pyruvate carboxylic moiety. Binding was also inhibited in the presence of free methyl 4,6-o-(carboxyethylidine)-beta-D-galactopyranoside. No protein resembling C-reactive protein (CRP) was obtained by calcium-dependent affinity chromatography of acute-phase guinea-pig serum on phosphorylcholine (PC)-Sepharose, and it not clear whether a counterpart of CRP exists in this species. Images Figure 1 Figure 2 PMID:3770806

  1. Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactobacillus [correction of Lactocobacillus] buchneri.


    Yildirim, Zeliha; Avşar, Yahya Kemal; Yildirim, Metin


    Buchnericin-LB adsorbs to gram-positive but not to gram-negative bacteria. The tested gram-positive bacteria were species of Lactobacillus, Pediococcus, Leuconostoc, Enterococcus, Lactococcus, Listeria, Bacillus, Staphylococcus; gram-negative bacteria belonged to the genera Salmonella, Escherichia, Yersinia and Pseudomonas. Buchnericin-LB adsorption depended on pH but not on time and temperature. Also some anions of salts and lipoteichoic acid reduced or inhibited its adsorption. Treatment of cells and cell walls of sensitive bacteria with detergents, organic solvents or enzymes did not affect subsequent binding of buchnericin-LB. Treatment with buchnericin-LB caused sensitive cells to lose high amounts of intracellular K+ ions and UV-absorbing materials and became more permeable to o-nitrophenol-beta-D-galactopyranoside. Buchnericin-LB (640-2560 AU/ml) decreased the colony forming units (99%) and absorbance values of Listeria monocytogenes and Bacillus cereus. These results indicate that the mode of action of buchnericin-LB is bactericidal and its lethal effect is very rapid.

  2. Enterobacter pulveris sp. nov., isolated from fruit powder, infant formula and an infant formula production environment.


    Stephan, Roger; Van Trappen, Stefanie; Cleenwerck, Ilse; Iversen, Carol; Joosten, Han; De Vos, Paul; Lehner, Angelika


    Six Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped isolates were obtained from fruit powder (n=3), infant formula (n=2) and an infant formula production environment (n=1) and investigated in a polyphasic taxonomic study. Comparative 16S rRNA gene sequence analysis combined with rpoB gene sequence analysis allocated the isolates to the family Enterobacteriaceae. The highest rpoB gene sequence similarities (91.2-95.8%) were obtained with Enterobacter helveticus, Enterobacter radicincitans, Enterobacter turicensis and Enterobacter sakazakii and the phylogenetic branch formed by these species was supported by a high bootstrap value. Biochemical data revealed that the isolates could be differentiated from their nearest neighbours by their ability to utilize melibiose, sucrose, D-arabitol, mucate and 1-O-methyl-alpha-galactopyranoside and their negative reactions for D-sorbitol utilization and the Voges-Proskauer test. On the basis of the phylogenetic analyses, DNA-DNA hybridization data, and unique physiological and biochemical characteristics, it is proposed that the isolates represent a novel species of the genus Enterobacter, Enterobacter pulveris sp. nov. The type strain is 601/05(T) (=LMG 24057(T)=DSM 19144(T)).

  3. Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker's yeast).

    PubMed Central

    Kundu, M; Basu, J; Ghosh, A; Chakrabarti, P


    The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin. Images Fig. 5. PMID:3128265

  4. Preparation and characterization of recombinant dolphin fish (Coryphaena hippurus) growth hormone.


    Paduel, A; Chapnik-Cohen, N; Gertler, A; Elizur, A


    Dolphin fish (Coryphaena hippurus) growth hormone (dfGH) cDNA encoding the mature protein was cloned in a pET11a expression vector and expressed in Escherichia coli BL21 cells upon induction with isopropyl-1-thio-beta-d-galactopyranoside as an insoluble protein. The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to homogeneity, as evidenced by SDS-PAGE. Gel filtration on a Superdex column under nondenaturing conditions and amino-terminal analysis showed the purified protein to be monomeric methionyl-dfGH. Binding assays of the (125)I-labeled dfGH to dolphin fish liver microsomal fraction resulted in high specific binding characterized by a K(a) of 0.77 nM(-1) and a B(max) of 285 fmol/mg microsomal fraction protein. The purified dfGH was capable of stimulating proliferation of FDC-P1-B9 cells transfected with rabbit growth hormone (GH) receptor. The maximal effect of dfGH was identical to that of human GH but their respective EC(50) values were 28 nM versus 0.095 nM.

  5. A 19F NMR Approach using Reporter Molecule Pairs to Assess β-Galactosidase in Human Xenograft Tumors in Vivo

    PubMed Central

    Yu, Jian-Xin; Kodibagkar, Vikram D.; Liu, Li; Mason, Ralph P.


    Gene therapy has emerged as a promising strategy for treatment of various diseases. However, widespread implementation is hampered by difficulties in assessing the success of transfection in the target tissue and the longevity of gene expression. Thus, there is increasing interest in the development of non-invasive in vivo reporter techniques to assay gene expression. We recently demonstrated the ability to detect β-galactosidase activity in stably transfected human prostate tumor xenografts in mice in vivo using 19F NMR. We now extend the studies to human MCF7 breast cancer cells growing as xenografts in nude mice. Moreover, by using two spectrally resolved reporters (o-fluoro-p-nitrophenyl-β-D-galactopyranoside and an isomer) two tumors could be interrogated simultaneously revealing lacZ transgene activity in a stably transfected tumor versus no activity in a wild type tumor. Most significantly hydrolytic activity observed by 19F NMR corresponded with differential activity in lacZ expressing tumors. PMID:18288788

  6. Surface activation of graphene oxide nanosheets by ultraviolet irradiation for highly efficient anti-bacterials

    NASA Astrophysics Data System (ADS)

    Veerapandian, Murugan; Zhang, Linghe; Krishnamoorthy, Karthikeyan; Yun, Kyusik


    A comprehensive investigation of anti-bacterial properties of graphene oxide (GO) and ultraviolet (UV) irradiated GO nanosheets was carried out. Microscopic characterization revealed that the GO nanosheet-like structures had wavy features and wrinkles or thin grooves. Fundamental surface chemical states of GO nanosheets (before and after UV irradiation) were investigated using x-ray photoelectron spectroscopy and ultraviolet photoelectron spectroscopy. Minimum inhibitory concentration (MIC) results revealed that UV irradiated GO nanosheets have more pronounced anti-bacterial behavior than GO nanosheets and standard antibiotic, kanamycin. The MIC of UV irradiated GO nanosheets was 0.125 μg ml-1 for Escherichia coli and Salmonella typhimurium, 0.25 μg ml-1 for Bacillus subtilis and 0.5 μg ml-1 for Enterococcus faecalis, ensuring its potential as an anti-infective agent for controlling the growth of pathogenic bacteria. The minimum bactericidal concentration of normal GO nanosheets was determined to be two-fold higher than its corresponding MIC value, indicating promising bactericidal activity. The mechanism of anti-bacterial action was evaluated by measuring the enzymatic activity of β-d-galactosidase for the hydrolysis of o-nitrophenol-β-d-galactopyranoside.

  7. Multiplex analysis of enzyme kinetics and inhibition by droplet microfluidics using picoinjectors.


    Sjostrom, Staffan L; Joensson, Haakan N; Svahn, Helene Andersson


    Enzyme kinetics and inhibition is important for a wide range of disciplines including pharmacology, medicine and industrial bioprocess technology. We present a novel microdroplet-based device for extensive characterization of the reaction kinetics of enzyme substrate inhibitor systems in a single experiment utilizing an integrated droplet picoinjector for bioanalysis. This device enables the scanning of multiple fluorescently-barcoded inhibitor concentrations and substrate conditions in a single, highly time-resolved experiment yielding the Michaelis constant (Km), the turnover number (kcat) and the enzyme inhibitor dissociation constants (ki, ki'). Using this device we determine Km and kcat for β-galactosidase and the fluorogenic substrate Resorufin β-D-galactopyranoside (RBG) to be 442 μM and 1070 s(-1), respectively. Furthermore, we examine the inhibitory effects of isopropyl-β-D-thiogalactopyranoside (IPTG) on β-galactosidase. This system has a number of potential applications, for example it could be used to screen inhibitors to pharmaceutically relevant enzymes and to characterize engineered enzyme variants for biofuels production, in both cases acquiring detailed information about the enzyme catalysis and enzyme inhibitor interaction at high throughput and low cost.

  8. Dependency of the regio- and stereoselectivity of intramolecular, ring-closing glycosylations upon the ring size

    PubMed Central

    Claude, Patrick; Lehmann, Christian


    Summary Phenyl 3,4,6-tri-O-benzyl-2-O-(3-carboxypropionyl)-1-thio-β-D-galactopyranoside (1) was condensed via its pentafluorophenyl ester 2 with 5-aminopentyl (4a), 4-aminobutyl (4b), 3-aminopropyl (4c) and 2-aminoethyl 4,6-O-benzylidene-β-D-glucopyranoside (4d), prepared from the corresponding N-Cbz protected glucosides 3a–d, to give the corresponding 2-[3-(alkylcarbamoyl)propionyl] tethered saccharides 5a–d. Intramolecular, ring closing glycosylation of the saccharides with NIS and TMSOTf afforded the tethered β(1→3) linked disaccharides 6a–c, the α(1→3) linked disaccharides 7a–d and the α(1→2) linked disaccharide 8d in ratios depending upon the ring size formed during glycosylation. No β(1→2) linked disaccharides were formed. Molecular modeling of saccharides 6–8 revealed that a strong aromatic stacking interaction between the aromatic parts of the benzyl and benzylidene protecting groups in the galactosyl and glucosyl moieties was mainly responsible for the observed regioselectivity and anomeric selectivity of the ring-closing glycosylation step. PMID:22238538

  9. Carotenoids, carotenoid esters, and anthocyanins of yellow-, orange-, and red-peeled cashew apples (Anacardium occidentale L.).


    Schweiggert, Ralf M; Vargas, Ester; Conrad, Jürgen; Hempel, Judith; Gras, Claudia C; Ziegler, Jochen U; Mayer, Angelika; Jiménez, Víctor; Esquivel, Patricia; Carle, Reinhold


    Pigment profiles of yellow-, orange-, and red-peeled cashew (Anacardium occidentale L.) apples were investigated. Among 15 identified carotenoids and carotenoid esters, β-carotene, and β-cryptoxanthin palmitate were the most abundant in peels and pulp of all samples. Total carotenoid concentrations in the pulp of yellow- and red-peeled cashew apples were low (0.69-0.73 mg/100g FW) compared to that of orange-peeled samples (2.2mg/100g FW). The color difference between the equally carotenoid-rich yellow and red colored samples indicated the presence of a further non-carotenoid pigment type in red peels. Among four detected anthocyanins, the major anthocyanin was unambiguously identified as 7-O-methylcyanidin 3-O-β-D-galactopyranoside by NMR spectroscopy. Red and yellow peel color was chiefly determined by the presence and absence of anthocyanins, respectively, while the orange appearance of the peel was mainly caused by increased carotenoid concentrations. Thus, orange-peeled fruits represent a rich source of provitamin A (ca. 124 μg retinol-activity-equivalents/100g pulp, FW).

  10. Metal-to-ligand charge-transfer: Applications to visual detection of β-galactosidase activity and sandwich immunoassay.


    Hu, Qiong; Ma, Kefeng; Mei, Yaqi; He, Minhui; Kong, Jinming; Zhang, Xueji


    In this work, we report a novel use of the distinctive metal-to-ligand charge-transfer (MLCT) absorption properties of the chromogenic Fe(BPDS)3(4-) (BPDS=bathophenanthroline disulfonic acid) reporter for the visual detection of β-galactosidase (β-Gal) activity and sandwich immunoassay. The enzymatic hydrolysis of the substrate p-aminophenyl-β-D-galactopyranoside can switch on the reduction of Fe(3+) to Fe(2+) and the subsequent complexation of Fe(2+) with the BPDS ligand to generate the Fe(BPDS)3(4-) reporter, leading to the appearance of the intense MLCT absorption band at 535nm and the colorless-to-red color change of the solution. Simply through a single step, the activity of β-Gal can be sensitively and selectively detected within the dynamic range of 0-220mUmL(-1), with a limit of detection (LOD) of 1.69mUmL(-1). This approach is applicable for the visual detection of β-Gal activities in the presence of complex human serum samples. Besides, when integrated with the sandwich immunoassay of carcinoembryonic antigen, a LOD of 1.16ngmL(-1) can be achieved. In light of its prominent simplicity and practicality, our MLCT-based approach holds great potential in diagnostic and analytical applications.

  11. A Novel Highly Thermostable Multifunctional Beta-Glycosidase from Crenarchaeon Acidilobus saccharovorans.


    Gumerov, Vadim M; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V


    We expressed a putative β-galactosidase Asac_1390 from hyperthermophilic crenarchaeon Acidilobus saccharovorans in Escherichia coli and purified the recombinant enzyme. Asac_1390 is composed of 490 amino acid residues and showed high sequence similarity to family 1 glycoside hydrolases from various thermophilic Crenarchaeota. The maximum activity was observed at pH 6.0 and 93°C. The half-life of the enzyme at 90°C was about 7 hours. Asac_1390 displayed high tolerance to glucose and exhibits hydrolytic activity towards cellobiose and various aryl glucosides. The hydrolytic activity with p-nitrophenyl (pNP) substrates followed the order pNP-β-D-galactopyranoside (328 U mg(-1)), pNP-β-D-glucopyranoside (246 U mg(-1)), pNP-β-D-xylopyranoside (72 U mg(-1)), and pNP-β-D-mannopyranoside (28 U mg(-1)). Thus the enzyme was actually a multifunctional β-glycosidase. Therefore, the utilization of Asac_1390 may contribute to facilitating the efficient degradation of lignocellulosic biomass and help enhance bioconversion processes.

  12. Oxidation increases mucin polymer cross-links to stiffen airway mucus gels.


    Yuan, Shaopeng; Hollinger, Martin; Lachowicz-Scroggins, Marrah E; Kerr, Sheena C; Dunican, Eleanor M; Daniel, Brian M; Ghosh, Sudakshina; Erzurum, Serpel C; Willard, Belinda; Hazen, Stanley L; Huang, Xiaozhu; Carrington, Stephen D; Oscarson, Stefan; Fahy, John V


    Airway mucus in cystic fibrosis (CF) is highly elastic, but the mechanism behind this pathology is unclear. We hypothesized that the biophysical properties of CF mucus are altered because of neutrophilic oxidative stress. Using confocal imaging, rheology, and biochemical measures of inflammation and oxidation, we found that CF airway mucus gels have a molecular architecture characterized by a core of mucin covered by a web of DNA and a rheological profile characterized by high elasticity that can be normalized by chemical reduction. We also found that high levels of reactive oxygen species in CF mucus correlated positively and significantly with high concentrations of the oxidized products of cysteine (disulfide cross-links). To directly determine whether oxidation can cross-link mucins to increase mucus elasticity, we exposed induced sputum from healthy subjects to oxidizing stimuli and found a marked and thiol-dependent increase in sputum elasticity. Targeting mucin disulfide cross-links using current thiol-amino structures such as N-acetylcysteine (NAC) requires high drug concentrations to have mucolytic effects. We therefore synthesized a thiol-carbohydrate structure (methyl 6-thio-6-deoxy-α-D-galactopyranoside) and found that it had stronger reducing activity than NAC and more potent and fast-acting mucolytic activity in CF sputum. Thus, oxidation arising from airway inflammation or environmental exposure contributes to pathologic mucus gel formation in the lung, which suggests that it can be targeted by thiol-modified carbohydrates.

  13. Evaluation of 36 Minitek tests and a new approach for identification of nonfermenters.


    Yabuuchi, E; Yamanaka, K; Ohyama, A


    Thirty-six Minitek (BBL Microbiology Systems, Cockeysville, Md.) tests were evaluated with 625 Kansai Medical University stock strains of 22 species and one group of nonfermentative gram-negative rods able to grow on ordinary peptone media. Among the 36 tests, 15 were selected because a clear-cut reaction was shown by all 625 Kansai Medical University strains. Of these 15 tests, 12 were further selected for routine use because they were regarded as useful for the identification of nonfermenters. The 12 tests were arranged into the following four groups: (i) lysine-arginine-ornithine, (ii) urea-ortho-nitrophenyl-beta-D-galactopyranoside-dextrose aerobic, (iii) maltose-xylose-starch, and (iv) esculin-nitrate reduction-indole. A new profile system for four digits, the Minitek Y-Y (Yabuuchi and Yamanaka) system, consisting of 64 numbers which represent each single species and 11 numbers which give two to four species, is herein proposed. The system was designed primarily for a less expensive identification of gram-negative rods already confirmed in a butt of either triple sugar iron or Kligler iron agar for their lack of ability to ferment dextrose. Among the 539 clinical isolates obtained from 3 hospitals, 511 strains identifiable by classical methods were also identified by the Minitek Y-Y system.

  14. [Development of chromogenic agar medium for isolation of enterohaemorrhagic Escherichia coli O26].


    Ikedo, M; Komatsu, O; Hara-Kudo, Y; Yamamoto, S; Kumagai, S


    Agar media for isolation of enterohaemorrhagic Escherichia coli (EHEC) have been developed primarily for E. coli O157, because this bacterium has most frequently caused EHEC infection. However, there have been few studies for isolation of other serotypes of EHEC, and media appropriate for isolation of such organisms, especially from food samples, are not yet available. Among such serotypes, E. coli O26 has often been isolated from clinical specimens from patients and animals, but not from food samples in outbreaks, because of lack of an appropriate method for isolation. In this study, we tried to develop a new chromogenic agar medium for selective isolation of E. coli O26 using the characteristics of E. coli O26. Fifteen strains of E. coli O26, 11 strains of E. coli O157 and 36 strains of other sero-types E. coli were tested for fermentation of rhamnose, cellobiose, dulcitol, salicin, raffinose, sorbitol, sucrose, lactose, mannitol, arabinose, maltose, xylose and glucose. Rhamnose was fermented by all E. coli strains except for E. coli O26. The other substrates were not effective for differentiating E. coli O26 from the other strains of E. coli. Thus the medium containing rhamnose and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, which is a substrate of beta-galactosidase specific to coliforms, produced a color of E. coli O26 colonies different from colors of the other bacteria. Furthermore, cefixime and sodium tellulite were added to the composition of the medium for gaining higher selectivity.

  15. Coarse-grained Simulations of Sugar Transport and Conformational Changes of Lactose Permease

    NASA Astrophysics Data System (ADS)

    Liu, Jin; Jewel, S. M. Yead; Dutta, Prashanta


    Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves sugar translocation, H+ transfer, as well as large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the transport of a β-D-galactopyranosyl-1-thio- β-D-galactopyranoside (TDG) molecule across a wild-type LacY during lactose/H+ symport process. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X-ray experiment. Protonation of Glu325 stabilizes the TDG and inward-facing conformation of LacY. Protonation of Glu269 induces a dramatic protein structural reorganization and causes the expulsion of TDG from LacY to both sides of the membrane. The structural changes occur primarily in the N-terminal domain of LacY. This work is supported by NSF Grants: CBET-1250107 and CBET -1604211.

  16. Preparation of monodisperse and size-controlled poly(ethylene glycol) hydrogel nanoparticles using liposome templates.


    An, Se Yong; Bui, Minh-Phuong Ngoc; Nam, Yun Jung; Han, Kwi Nam; Li, Cheng Ai; Choo, Jaebum; Lee, Eun Kyu; Katoh, Shigeo; Kumada, Yoichi; Seong, Gi Hun


    Liposomes were used as templates to prepare size-controlled and monodisperse poly(ethylene glycol) (PEG) hydrogel nanoparticles. The procedure for the preparation of PEG nanoparticles using liposomes consists of encapsulation of photopolymerizable PEG hydrogel solution into the cavity of the liposomes, extrusion through a membrane with a specific pore size, and photopolymerization of the contents inside the liposomes by UV irradiation. The size distributions of the prepared particles were 1.32+/-0.16 microm (12%), 450+/-62 nm (14%), and 94+/-12 nm (13%) after extrusion through membrane filters with pore sizes of 1 microm, 400 nm, and 100 nm, respectively. With this approach, it is also possible to modify the surface of the hydrogel nanoparticles with various functional groups in a one-step procedure. To functionalize the surface of a PEG nanoparticle, methoxy poly(ethylene glycol)-aldehyde was added as copolymer to the hydrogel-forming components and aldehyde-functionalized PEG nanoparticles could be obtained easily by UV-induced photopolymerization, following conjugation with poly-L-lysine-FITC through amine-aldehyde coupling. The prepared PEG particles showed strong fluorescence from FITC on the edge of the particles using confocal microscopy. The immobilization of biomaterials such as enzymes in hydrogel particles could be performed with loading beta-galactosidases during the hydration step for liposome preparation without additional procedures. The resorufin produced by applying resorufin beta-D-galactopyranoside as the substrate showed the fluorescence under the confocal microscopy.

  17. Comparison of the recoveries of Escherichia coli and total coliforms from drinking water by the MI agar method and the U.S. Environmental Protection Agency-approved membrane filter method.

    PubMed Central

    Brenner, K P; Rankin, C C; Sivaganesan, M; Scarpino, P V


    Drinking water regulations under the Final Coliform Rule require that total coliform-positive drinking water samples be examined for the presence of Escherichia coli or fecal coliforms. The current U.S. Environmental Protection Agency-approved membrane filter (MF) method for E. coli requires two media, an MF transfer, and a total incubation time of 28 h. A newly developed MF method, the MI agar method, containing indoxyl-beta-D-glucuronide and 4-methylumbelliferyl-beta-D-galactopyranoside for the simultaneous detection of E. coli and total coliforms, respectively, by means of their specific enzyme reactions, was compared with the approved method by the use of wastewater-spiked tap water samples. Overall, weighted analysis of variance (significance level, 0.05) showed that the new medium recoveries of total coliforms and E. coli were significantly higher than those of mEndo agar and nutrient agar plus MUG (4-methylumbelliferyl-beta-D-glucuronide), respectively, and the background counts were significantly lower than those of mEndo agar (< 5%). Generally, the tap water source, overall chlorine level, wastewater source, granular activated carbon treatment of the tap water, and method of grouping data by E. coli count for statistical analysis did not affect the performance of the new medium. PMID:8572697

  18. Production, purification, and characterization of a novel galactose oxidase from Fusarium acuminatum.


    Alberton, Dayane; Silva de Oliveira, Luciana; Peralta, Rosane Marina; Barbosa-Tessmann, Ione Parra


    Extra-cellular production of a novel galactose oxidase from Fusarium acuminatum using submerged fermentation was studied. Glucose (1.0% w/v) was used as the sole carbon source. Maximum galactose oxidase production (approximately 4.0 U/ml) was obtained when fermentation was carried out at 25 degrees C, with orbital shaking (100 rpm) and an initial medium of pH 7.0, for 96 h, using a 2% (v/v) inoculum made from a homogenized four-day-old liquid culture, in the presence of copper, manganese, and magnesium. The enzyme was purified by one-step affinity chromatography, with a recovery of 42% of the initial activity. The purified enzyme ran as a single band of 66 kDa in SDS-PAGE. Optimal pH and temperature for the enzyme activity were 8.0 and 30 degrees C, respectively. The enzyme was thermoinactivated at temperatures above 60 degrees C. The purified enzyme was active toward various substrates, including galactose, dihydroxyacetone, guar gum, lactose, melibiose, methyl-galactopyranoside, and raffinose. SDS was an inhibitor but EDTA, Tween 80, NH(4)(+), Na(+), Mg(2+), K(+), and glycerol were not. The Michaelis-Menten constant (K(m)) for galactose was estimated to be 16.2 mM, while maximal velocity (V(max)) was 0.27 micromol of H(2)O(2) . ml(-1) . min(-1).

  19. Structure of the β-Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

    PubMed Central

    Vian, Alejandro; Carrascosa, Alfonso V.; García, José L.; Cortés, Estrella


    The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (Mr, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a Km value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C. PMID:9603833

  20. A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.


    Obayashi, Yusuke; Iino, Ryota; Noji, Hiroyuki


    Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-d-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.

  1. Phytochemical Profiling and Evaluation of Pharmacological Activities of Hypericum scabrum L.


    Jiang, Lan; Numonov, Sodik; Bobakulov, Khayrulla; Qureshi, Muhammad Nasimullah; Zhao, Haiqing; Aisa, Haji Akber


    Phytochemical investigations of ethyl acetate-soluble part of the aerial part of Hypericum scabrum L. delivered eight pure phenolic compounds 1-8. The pure compounds were identified through physico-chemical, NMR (1D, 2D) and mass spectrometric studies as: 3-8''-bisapigenin (1), quercetin (2), quercetin-3-O-α-l-arabinofuranoside (3), quercetin-3-O-α-l-rhamnoside (4), quercetin-3-O-β-d-glucopyranoside (5), quercetin-3-O-β-d-galactopyranoside (6), (-)-epicatechin (7), (+)-catechin (8). Total polyphenolic compounds and total flavonoids contents were determined in the extract as 0.107 mg∙mg-1 and 0.023 mg∙mg-1 of the dried extract, respectively. Antioxidant activity using DPPH free radical scavenging assay delivered very strong activity for compounds 2 and 5, 6 and crude extract 10. Protein tyrosine phosphatase 1B (PTP-1B) inhibition experiment of isolated compounds and crude extracts resulted in significant inhibition activity for samples 2, 7a, 8a, 11 and 12 with IC50 values ranging from 1.57 to 2.91 µM. Antimicrobial activity of the pure compounds and extracts produced average results against Staphylococcus aureus, Escherichia coli and Candida albicans strains. From our literature survey, it appears that all pure compounds except 2 were isolated and reported for the first time in H. scabrum.

  2. Flavonoid Detection in Hydroethanolic Extract of Pouteria torta (Sapotaceae) Leaves by HPLC-DAD and the Determination of Its Mutagenic Activity

    PubMed Central

    Costa, Daryne L.M.G.; Rinaldo, Daniel; Varanda, Eliana A.; de Sousa, Juliana F.; Nasser, Ana L.M.; Silva, Ana C.Z.; Baldoqui, Débora C.; Vilegas, Wagner


    Abstract It is well known that phytotherapy has grown in popularity in recent years. Because a drug cannot be administered without ensuring its effectiveness and safety, the standardization and regulation of phytotherapeutic drugs are required by the global market and governmental authorities. This article describes a simple and reliable high-performance liquid chromatography–diode array detection analysis method for the simultaneous detection of myricetin-3-O-β-D-galactopyranoside, myricetin-3-O-α-L-arabinopyranoside, and myricetin-3-O-α-L-rhaminopyranoside present in the hydroethanolic extract (ethanol/H2O, 7:3, v/v) of Pouteria torta. The mutagenic activity of the extract was evaluated on Salmonella typhimurium and by an in vivo micronucleus test on the peripheral blood cells of Swiss mice. The linearity, sensitivity, selectivity, repeatability, accuracy, and precision of the assay were evaluated. The analytical curves were linear and exhibited good repeatability (with a deviation of less than 5%) and demonstrated good recovery (within the 83–107% range). The results demonstrate that the hydroethanolic extract exhibited a mutagenic activity in both assays, suggesting caution in the use of this plant in folk medicine. PMID:25055245

  3. Detection of Escherichia coli in drinking water using T7 bacteriophage-conjugated magnetic probe.


    Chen, Juhong; Alcaine, Samuel D; Jiang, Ziwen; Rotello, Vincent M; Nugen, Sam R


    In this study, we demonstrate a bacteriophage (phage)-based magnetic separation scheme for the rapid detection of Escherichia coli (E. coli) in drinking water. T7 phage is a lytic phage with a broad host range specificity for E. coli. Our scheme was as follows: (1) T7 bacteriophage-conjugated magnetic beads were used to capture and separate E. coli BL21 from drinking water; (2) subsequent phage-mediated lysis was used to release endemic β-galactosidase (β-gal) from the bound bacterial cells; (3) the release of β-gal was detected using chlorophenol red-β-d-galactopyranoside (CRPG), a colorimetric substrate which changes from yellow to red in the presence of β-gal. Using this strategy, we were able to detect E. coli at a concentration of 1 × 10(4) CFU·mL(-1) within 2.5 h. The specificity of the proposed magnetic probes toward E. coli was demonstrated against a background of competing bacteria. By incorporating a pre-enrichment step in Luria-Bertani (LB) broth supplemented with isopropyl β-d-thiogalactopyranoside (IPTG), we were able to detect 10 CFU·mL(-1) in drinking water after 6 h of pre-enrichment. The colorimetric change can be determined either by visual observation or with a reader, allowing for a simple, rapid quantification of E. coli in resource-limited settings.

  4. Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

    PubMed Central

    Simons, G; Nijhuis, M; de Vos, W M


    Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate. Images PMID:8349556

  5. Chemical profiling of anti-hepatocellular carcinoma constituents from Caragana tangutica Maxim. by off-line semi-preparative HPLC-NMR.


    Yang, Xinzhou; Huang, Mi; Cai, Jinyan; Lv, Dan; Lv, Jingnan; Zheng, Sijian; Ma, Xinhua; Zhao, Ping; Wang, Qiang


    An EtOAc fraction from the roots of Caragana tangutica Maxim. (CTEA) displayed promising anti-hepatocellular carcinoma (HCC) activity during screening of a traditional Chinese ethnic herb library against HepG2 and Hep3B cell lines. HPLC-based activity profiling of CTEA by combination of MS-guided large-scale semi-preparative HPLC and NMR methods led to the identification of a new pterocarpan glycoside, (-)-maackiain 3-O-6'-O-methyl malonyl-β-d-glucopyranoside (1), together with three known pterocarpan glycosides, (-)-maackiain 3-O-β-d-glucopyranoside (2), 3-O-6'-O-acrylyl-β-d-galactopyranoside (3), and (-)-maackiain 3-O-6'-O-acetyl-β-d-glucopyranoside (4). Compound 1 was isolated during a drug discovery programme aimed at identifying new anti-HCC leads from a natural product library. Anti-HCC study showed that all four compounds exhibited cytotoxic activity with IC50 values range of 29.1-53.5 μg/mL against HepG2 and Hep3B cell lines.

  6. Double sugar-tyrosine medium improves O-1 phage Salmonella screening.

    PubMed Central

    Thaller, M C; Dainelli, B; Berlutti, F; Schippa, S; Fontana, C; Pezzi, R


    A modification of the procedure for O-1 phage Salmonella screening is presented. The novel method is based on the use of two media, i.e., a new medium (double sugar-tyrosine [DST]), which permits the combination of adonitol and sucrose fermentation and tyrosine clearing tests, and the previously described o-nitrophenyl-beta-D-galactopyranoside urease indole medium. In comparative trials, the new procedure and the conventional one were used to screen for Salmonella isolates from 553 lactose-negative strains of members of the family Enterobacteriaceae. The O-1 phage test, performed on DST medium, recognized the same number of phage-susceptible Salmonella strains as did the standardized method; however, it permitted the correct identification of a greater number of phage-resistant strains for discard (95.6 versus 85.3%). In particular, DST medium presented a higher efficacy than triple sugar iron agar (which is the corresponding medium in the reference procedure) in correctly identifying phage-negative cultures for discard (69.1 versus 28.5%). PMID:1537932

  7. Anti-inflammatory activity of Pistacia khinjuk in different experimental models: isolation and characterization of its flavonoids and galloylated sugars.


    Esmat, Ahmed; Al-Abbasi, Fahad A; Algandaby, Mardi M; Moussa, Ashaimaa Y; Labib, Rola M; Ayoub, Nahla A; Abdel-Naim, Ashraf B


    The present study aimed at isolating and elucidating the structure of the main components of Pistacia khinjuk L. and exploring its potential anti-inflammatory effect in different experimental models. The extract was evaluated for anti-inflammatory activity by measuring paw volume in three experimental models. Then, prostaglandin E₂ (PGE₂) level, ear edema, tissue myeloperoxidase (MPO) activity, histopathology, nitric oxide (NO) level, and tumor necrosis factor-α (TNF-α) level were assessed. Seven phenolic compounds, mainly flavonoids and galloylated compounds, were isolated from the aqueous methanol extract: gallic acid (1), methyl gallate (2), quercetin-3-O-β-D-⁴C₁-galactopyranoside (hyperin) (3), myricetin-3-O-α-L-¹C₄-rhamnopyranoside (myricitrin) (4), 1,6-digalloyl-β-D-glucose (5), 1,4-digalloyl-β-D-glucopyranoside (6), and 2,3-di-O-galloyl-(α/β)-⁴C₁-glucopyranose (nilocitin) (7). The anti-inflammatory activity was evidenced by decreased carrageenan-induced rat paw edema and PGE₂ elevation. In the croton oil-induced ear edema model, MPO activity was significantly inhibited, and inflammatory histopathological changes were ameliorated. In the rat air pouch model, NO generation and TNF-α release were significantly inhibited. The isolation and nuclear magnetic resonance spectral data of compound 6 from the genus Pistacia are revealed for the first time. Also, P. khinjuk L. aqueous methanol extract possesses anti-inflammatory activity in several experimental models.

  8. [Molecular cloning, prokaryotic expression and double-antibody sandwich ELISA development of 17β-hsd10 in mouse].


    Liu, Chuanzhi; Niu, Yingying; Chen, Yuan'an; Wu, Cheng; Yu, Yuanhua


    We expressed 17-hydroxysteroid dehydrogenase10 (17β-hsd10) recombinant protein, prepared anti-17β- hsd10 polyclonal antibodies and established sandwich enzyme linked immunosorbent assay (ELISA) test for detection of 17β-hsd10. RT-PCR was used to get the gene of 17β-hsd10 of mouse liver, and a prokaryotic protein expression system pET 15b-17β-hsd10/Escherichia coli BL21 (DE3) which induced with isopropyl-1-thio-β-galactopyranoside (IPTG) for recombinant protein expression was constructed subsequently. The target protein purified using His-Binding-resin column was used to immunize BALB/c mice and rabbits, serum total IgGs from immunized animals were purified by ammonium sulfate precipitation method. We established a Double-antibody Sandwich enzyme linked immunosorbent assay about 17β-hsd10 using the two antibodies we prepared. We got the concentration of 1.5 mg/mL of 17β-hsd10 protein with molecular weight of 29.5 kDa, and polyclonal antibodies from mouse and rabbit with the tite 1.25 x 10(4) and 2.5 x 10(4) respectively. The concentration of 0.1 g/mL of 17β-hsd10 can be detected by the Double-antibody Sandwich ELISA we established, and the assay was sensitive and specific. It can be widely used in clinical and experimental study.

  9. Synthesis of azido-functionalized carbohydrates for the design of glycoconjugates.


    Cecioni, Samy; Goyard, David; Praly, Jean-Pierre; Vidal, Sébastien


    As carbohydrates play a major role in numerous biological processes through their interactions with lectins and also appear as one of the most crucial post-translational modifications of proteins, chemists have developed several approaches for the design of glycoconjugates based on a series of conjugation methodologies. The recent development of copper(I)-catalyzed azide-alkyne cycloaddition (CuACC) paved the way to a novel conjugation strategy in which azido-functionalized carbohydrate derivatives can be readily connected to alkyne-functionalized (bio)molecules. This so-called "click chemistry" methodology has now found numerous applications both in chemistry and biology. The azido moiety can be introduced either directly at the anomeric carbon of the carbohydrate derivative, or attached to a spacer arm. We describe here the syntheses of 2,3,4,6-tetra-O-acetyl-β-D: -glucopyranosyl azide as well as 1-azido-3,6-dioxaoct-8-yl 2,3,4,6-tetra-O-acetyl-β-D: -galactopyranoside and 1-azido-3,6-dioxaoct-8-yl 2,3,6,2',3',4',6'-hepta-O-acetyl-β-D: -lactoside. These molecules can then be used in the construction of glycoconjugates to find applications in chemical biology.

  10. Studies on lectins. XLIX. The use of glycosyl derivatives of Dextran T-500 for affinity electrophoresis of lectins.


    Cerovský, V; Tichá, M; Horejsi, V; Kocourek, J


    p-Aminophenyl glycosides and glycosylamines were coupled to periodate oxidized Dextran T-500 either directly or through an epsilon-aminocaproic acid spacer. The new glycosylated derivatives of dextran specifically precipitate lectins having the appropriate carbohydrate specificity, and thus were used in the preparation of affinity gels for affinity electrophoresis of lectins. The apparent strength of interaction of several lectins with carbohydrate residues immobilized in this way was less than with carbohydrates immobilized in O-glycosyl polyacrylamide copolymers. The presence of epsilon-aminocaproic spacer had no effect on the strength of interaction. The advantages of this type of macromolecular derivative of the ligand for affinity electrophoresis and some differences between the glycosylated dextrans and O-glycosyl polyacrylamide copolymers are discussed. Dextrans containing bound p-aminophenyl alpha-D-mannopyranoside and p-aminophenyl alpha-D-glucopyranoside were used to study the binding properties of concanavalin A and the lectin from Lathyrus sativus seeds. For the investigation of interaction of lectins from Ricinus communis and Glycine soja seeds, dextran derivatives containing bound p-aminophenyl alpha- and beta-D-galactopyranosides and alpha- and beta-D-galactopyranosylamines were used.

  11. A lectin with some unique characteristics from the samta tomato.


    Wang, H; Ng, T B


    A lectin, with a molecular mass of 79 kDa, and with specificity toward rhamnose and O-nitrophenyl-beta-D-galactopyranoside, was isolated from samta tomato fruits. The procedure entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel and CM-cellulose. The lectin was stable up to 70 degrees C. The lectin activity was potentiated by NaOH solutions (25-100 mM), but was reduced by 50 and 100 mM HCl solutions. The activity of the lectin was reduced in the presence of CaCl(2), MgCl(2) and ZnCl(2), but was potentiated by 5 and 10 mM AlCl(3) solutions. The lectin stimulated the mitogenic response in mouse splenocytes and inhibited human immunodeficiency virus-1 reverse transcriptase with an IC(50) of 6.2 microM.

  12. Colorimetric Detection of Escherichia coli Based on the Enzyme-Induced Metallization of Gold Nanorods.


    Chen, Juhong; Jackson, Angelyca A; Rotello, Vincent M; Nugen, Sam R


    A novel enzyme-induced metallization colorimetric assay is developed to monitor and measure beta-galactosidase (β-gal) activity, and is further employed for colorimetric bacteriophage (phage)-enabled detection of Escherichia coli (E. coli). This assay relies on enzymatic reaction-induced silver deposition on the surface of gold nanorods (AuNRs). In the presence of β-gal, the substrate p-aminophenyl β-d-galactopyranoside is hydrolyzed to produce p-aminophenol (PAP). Reduction of silver ions by PAP generates a silver shell on the surface of AuNRs, resulting in the blue shift of the longitudinal localized surface plasmon resonance peak and multicolor changes of the detection solution from light green to orange-red. Under optimized conditions, the detection limit for β-gal is 128 pM, which is lower than the conventional colorimetric assay. Additionally, the assay has a broader dynamic range for β-gal detection. The specificity of this assay for the detection of β-gal is demonstrated against several protein competitors. Additionally, this technique is successfully applied to detect E. coli bacteria cells in combination with bacteriophage infection. Due to the simplicity and short incubation time of this enzyme-induced metallization colorimetric method, the assay is well suited for the detection of bacteria in low-resource settings.

  13. Determination of selenium urinary metabolites by high temperature liquid chromatography-inductively coupled plasma mass spectrometry.


    Terol, A; Ardini, F; Basso, A; Grotti, M


    The coupling of high temperature liquid chromatography (HTLC) and inductively coupled plasma mass spectrometry (ICPMS) for the determination of selenium metabolites in urine samples is reported for the first time. In order to achieve "ICPMS-friendly" chromatographic conditions, the retention on a graphite stationary phase of the major selenium urinary metabolites using only plain water with 2% methanol as the mobile phase was investigated. Under the optimal conditions (T=80°C, Ql=1.2 mL min(-1)), methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactopyranoside (selenosugar 1), methyl 2-acetamido-2-deoxy-1-seleno-β-d-glucosopyranoside (selenosugar 2) and trimethylselenonium ion were efficiently separated in less than 7 min, without any interferences due to other common selenium species (selenite, selenate, selenocystine and selenomethionine) or detectable effect of the urine matrix. The limits of detection were 0.3-0.5 ng Se mL(-1), and the precision of the analytical procedure was better than 3% (RSD%, n=5). The HTLC-ICPMS method was applied to the analysis of urine samples from two volunteers before and after ingestion of Brazil nuts or selenium supplements. The developed procedure proved to be adequate for the analytical task, providing results consistent with previous studies.

  14. Purification and properties of beta-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller.

    PubMed Central

    Calvo, P; Reglero, A; Cabezas, J A


    1. A beta-N-acetylhexosaminidase was purified 330-fold from the digestive gland of the terrestrial mollusc Helicella ericetorum Müller. 2. Its pH optimum is 4.5 for both beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities in two buffer solutions; it is fully stable at 37 degrees C for 2h in the pH range 3.8--4.6 and shows one isoelectric point (pH 4.83). 3. The estimated mol.wt. is between 120,000 and 145,000. 4. The enzyme shows an endo-beta-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulphate, chitin and hyaluronic acid. 5. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. 6. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and p-nitrophenyl 2-acetamide-2-deoxy-beta-D-galactopyranoside are 0.43 mM, 30.1 micronmol of p-nitrophenol/min per mg and 0.19 mM, 8.6 micronmol of p-nitrophenol/min per mg respectively. 7. It is inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. 8. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase activities are catalysed by the enzyme at the same active site. PMID:33660

  15. Structure of the lac operon galactoside acetyltransferase.


    Wang, Xing-Guo; Olsen, Laurence R; Roderick, Steven L


    The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.

  16. A transgenic mouse for imaging caspase-dependent apoptosis within the skin.


    Khanna, Divya; Hamilton, Christin A; Bhojani, Mahaveer S; Lee, Kuei C; Dlugosz, Andrej; Ross, Brian D; Rehemtulla, Alnawaz


    Apoptosis is an essential process for the maintenance of normal physiology. The ability to noninvasively image apoptosis in living animals would provide unique insights into its role in normal and disease processes. Herein, a recombinant reporter consisting of beta-galactosidase gene flanked by two estrogen receptor regulatory domains and intervening Asp-Glu-Val-Glu sequences was constructed to serve as a tool for in vivo assessment of apoptotic activity. The results demonstrate that when expressed in its intact form, the hybrid reporter had undetectable beta-galactosidase activity. Caspase 3 activation in response to an apoptotic stimulus resulted in cleavage of the reporter, and thereby reconstitution of beta-galactosidase activity. Enzymatic activation of the reporter during an apoptotic event enabled noninvasive measurement of beta-galactosidase activity in living cells, which correlated with traditional measures of apoptosis in a dose- and time-dependent manner. Using a near-infrared fluorescent substrate of beta-galactosidase (9H-{1,3-dichloro-9,9-dimethylacridin-2-one-7-yl} beta-D-galactopyranoside), noninvasive in vivo imaging of apoptosis was achieved in a xenograft tumor model in response to proapoptotic therapy. Finally, a transgenic mouse model was developed expressing the ER-LACZ-ER reporter within the skin. This reporter and transgenic mouse could serve as a unique tool for the study of apoptosis in living cells and animals, especially in the context of skin biology.

  17. Isolation of antioxidant phytoconstituents from the seeds of Lens culinaris Medik.


    Jameel, Mohammad; Ali, Abuzer; Ali, Mohammed


    Lens culinaris Medik (Leguminosae) is an annual, bushy and herbaceous plant cultivated globally for its edible seeds. A methanolic extract of the seeds contained four new antioxidant compounds, namely β-sitosteryl-3-(2'-n-eicosanyloxy)-benzoate (3), n-octadec-9-enoyl-1-β-D-glucurano-pyranoside (4) α-D-galactopyranosyl-(6 → 1')-α-D-galactopyranosyl-(6' → 1″)-α-D-galactopyranosyl-(6″ → 1‴)-α-d-galactopyranoside (5) and benzoyl-O-α-D-glucopyranosyl-(2a → 1b)-O-α-D-glucopyranosyl-(2b → 1c)-O-α-D-glucopyranosyl-(6c → 1d)-O-α-D-glucopyranosyl-(6d → 1e)-O-α-D-gluco-pyranoside (6) along with two known compounds n-heptadecanyl n-octadec-9-enoate (1) and β-sitosterol (2) on the basis of chromatographic and spectral data analytical techniques. Compound 3 showed significant antioxidant activity compared to compounds 4, 5, and 6.

  18. Computational analysis of human N-acetylgalactosamine-6-sulfate sulfatase enzyme: an update in genotype-phenotype correlation for Morquio A.


    Olarte-Avellaneda, Sergio; Rodríguez-López, Alexander; Alméciga-Díaz, Carlos Javier; Barrera, Luis Alejandro


    Mucopolysaccharidosis IV A (MPS IV A) is a lysosomal storage disease produced by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) enzyme. Although genotype-phenotype correlations have been reported, these approaches have not enabled to establish a complete genotype-phenotype correlation, and they have not considered a ligand-enzyme interaction. In this study, we expanded the in silico evaluation of GALNS mutations by using several bioinformatics tools. Tertiary GALNS structure was modeled and used for molecular docking against galactose-6-sulfate, N-acetylgalactosamine-6-sulfate, keratan sulfate, chondroitin-6-sulfate, and the artificial substrate 4-methylumbelliferyl-β-D-galactopyranoside-6-sulfate. Furthermore, we considered the evolutionary residue conservation, change conservativeness, position within GALNS structure, and the impact of amino acid substitution on the structure and function of GALNS. Molecular docking showed that amino acids involved in ligand interaction correlated with those observed in other human sulfatases, and mutations within the active cavity reduced affinity of all evaluated ligands. Combination of several bioinformatics approaches allowed to explaine 90% of the missense mutations affecting GALNS, and the prediction of the phenotype for another 21 missense mutations. In summary, we have shown for the first time a docking evaluation of natural and artificial ligands for human GALNS, and proposed an update in genotype-phenotype correlation for Morquio A, based on the use of multiple parameters to predict the disease severity.

  19. Structural studies of the carbohydrate moieties of lectins from potato (Solanum tuberosum) tubers and thorn-apple (Datura stramonium) seeds

    PubMed Central

    Ashford, David; Desai, Nila N.; Allen, Anthony K.; Neuberger, Albert; O'Neill, Malcolm A.; Selvendran, Robert R.


    1. Methylation analysis of potato (Solanum tuberosum) lectin and thorn-apple (Datura stramonium) lectin confirmed previous conclusions that both glycoproteins contained high proportions of l-arabinofuranosides and lesser amounts of d-galactopyranosides. The arabinofuranosides are present in both lectins as short unbranched chains containing 1→2- and 1→3-linkages, which are known to be linked to hydroxyproline. Galactopyranosides are present as monosaccharides, which are known to be attached to serine, in potato lectin and as both the monosaccharide and the 1→3-linked disaccharide in Datura lectin. 2. Alkaline digestion of potato lectin and subsequent separation of the components by gel filtration led to the isolation of four fractions corresponding to the mono-, di-, tri- and tetra-arabinosides of hydroxyproline. The latter two fractions accounted for over 70% of the total hydroxyproline. 3. Methylation analysis was used to show that the triarabinoside contained only 1→2-linkages between sugars, but that the tetra-arabinoside contained both 1→2- and 1→3-linkages. Direct-insertion mass spectrometry of these compounds using electron impact and chemical ionization, in a comparison with other known structural patterns, was used to determine the sequences of the sugars, which were Araƒ1→2Araƒ1→2Araƒ1→Hyp and Araƒ1→3Araƒ1→2Araƒ1→2Araƒ 1→Hyp. 4. On the basis of optical rotation it had previously been suggested [Allen, Desai, Neuberger & Creeth (1978) Biochem. J. 171, 665–674] that all the arabinose of potato lectin was present as the β-l-furanoside. However, measurement of the optical rotations of the hydroxyprolyl arabinosides showed that whereas the diarabinoside had a molar rotation ([m]) value close to that predicted, the triarabinoside was more dextrorotatory and the tetra-arabinoside was less dextrorotatory than expected. Possible explanations for these findings are that, although the di- and tri-arabinosides contain exclusively

  20. A galactose-functionalized dendritic siRNA-nanovector to potentiate hepatitis C inhibition in liver cells

    NASA Astrophysics Data System (ADS)

    Lakshminarayanan, Abirami; Reddy, B. Uma; Raghav, Nallani; Ravi, Vijay Kumar; Kumar, Anuj; Maiti, Prabal K.; Sood, A. K.; Jayaraman, N.; Das, Saumitra


    A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated region (UTR) of HCV RNA using a liver-targeted dendritic nano-vector functionalized with a galactopyranoside ligand (DG). Physico-chemical characterization revealed finer details of complexation of DG with siRNA, whereas molecular dynamic simulations demonstrated sugar moieties projecting ``out'' in the complex. Preferential delivery of siRNA to the liver was achieved through a highly specific ligand-receptor interaction between dendritic galactose and the asialoglycoprotein receptor. The siRNA-DG complex exhibited perinuclear localization in liver cells and co-localization with viral proteins. The histopathological studies showed the systemic tolerance and biocompatibility of DG. Further, whole body imaging and immunohistochemistry studies confirmed the preferential delivery of the nucleic acid to mice liver. Significant decrease in HCV RNA levels (up to 75%) was achieved in HCV subgenomic replicon and full length HCV-JFH1 infectious cell culture systems. The multidisciplinary approach provides the `proof of concept' for restricted delivery of therapeutic siRNAs using a target oriented dendritic nano-vector.A RNAi based antiviral strategy holds the promise to impede hepatitis C viral (HCV) infection overcoming the problem of emergence of drug resistant variants, usually encountered in the interferon free direct-acting antiviral therapy. Targeted delivery of siRNA helps minimize adverse `off-target' effects and maximize the efficacy of therapeutic response. Herein, we report the delivery of siRNA against the conserved 5'-untranslated

  1. Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

    NASA Astrophysics Data System (ADS)

    Liu, Shuang

    protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands

  2. Mechanistic evaluation of MelA α-galactosidase from Citrobacter freundii: a family 4 glycosyl hydrolase in which oxidation is rate-limiting.


    Chakladar, Saswati; Cheng, Lydia; Choi, Mary; Liu, James; Bennet, Andrew J


    The MelA gene from Citrobacter freundii, which encodes a glycosyl hydrolase family 4 (GH4) α-galactosidase, has been cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the hydrolysis of phenyl α-galactosides via a redox elimination-addition mechanism involving oxidation of the hydroxyl group at C-3 and elimination of phenol across the C-1-C-2 bond to give an enzyme-bound glycal intermediate. For optimal activity, the MelA enzyme requires two cofactors, NAD(+) and Mn(2+), and the addition of a reducing agent, such as mercaptoethanol. To delineate the mechanism of action for this GH4 enzyme, we measured leaving group effects, and the derived β(lg) values on V and V/K are indistinguishable from zero (-0.01 ± 0.02 and 0.02 ± 0.04, respectively). Deuterium kinetic isotope effects (KIEs) were measured for the weakly activated substrate phenyl α-D-galactopyranoside in which isotopic substitution was incorporated at C-1, C-2, or C-3. KIEs of 1.06 ± 0.07, 0.91 ± 0.04, and 1.02 ± 0.06 were measured on V for the 1-(2)H, 2-(2)H, and 3-(2)H isotopic substrates, respectively. The corresponding values on V/K were 1.13 ± 0.07, 1.74 ± 0.06, and 1.74 ± 0.05, respectively. To determine if the KIEs report on a single step or on a virtual transition state, we measured KIEs using doubly deuterated substrates. The measured (D)V/K KIEs for MelA-catalyzed hydrolysis of phenyl α-D-galactopyranoside on the dideuterated substrates, (D)V/K((3-D)/(2-D,3-D)) and (D)V/K((2-D)/(2-D,3-D)), are 1.71 ± 0.12 and 1.71 ± 0.13, respectively. In addition, the corresponding values on V, (D)V((3-D)/(2-D,3-D)) and (D)V((2-D)/(2-D,3-D)), are 0.91 ± 0.06 and 1.01 ± 0.06, respectively. These observations are consistent with oxidation at C-3, which occurs via the transfer of a hydride to the on-board NAD(+), being concerted with proton removal at C-2 and the fact that this step is the first irreversible step for the MelA α-galactosidase-catalyzed reactions of aryl

  3. Bioactive compounds of Eriocaulon sieboldianum blocking proliferation and inducing apoptosis of HepG2 cells might be involved in Aurora kinase inhibition.


    Fan, Yanhua; Lu, Hongyuan; Ma, Hongda; Feng, Fan; Hu, Xiaolong; Zhang, Qiao; Wang, Jian; Xu, Yongnan; Zhao, Qingchun


    Eriocaulon sieboldianum (Sieb. & Zucc. ex Steud.) is an edible and medicinal plant used in traditional Chinese medicine. Often in combination with other herbs, it is processed into healthcare beverages for expelling wind-heat, protecting eyes, and reducing blood lipids. Besides, its water decoction together with other herbs has been utilized to treat cancer in China. However, the active ingredients and the precise cellular mechanisms of E. sieboldianum remain to be elucidated. The Aurora kinase family plays critical roles in the regulation of cell division and has attracted great attention to the identification of small-molecule Aurora kinase inhibitors for potential treatment of cancer. A molecular docking study was employed for docking of the most bioactive compounds. Hispidulin (HPDL) and quercetin-3-O-(6''-O-galloyl)-β-D-galactopyranoside (QGGP) were singled out as potent inhibitors of Aurora kinase. Their inhibitory activity towards Aurora kinase was further confirmed by the obvious decrease in autophosphorylation of Aurora-A (Thr288) and Aurora-B (Thr232). Moreover, the induction of cell cycle arrest in HepG2 cells and the suppressed phosphorylation of histone H3 were also consistent with the inhibition of Aurora kinase. The data indicate that the E. sieboldianum extract and its two active compounds, HPDL and QGGP, could effectively induce apoptosis via p53, MAPKs and the mitochondrial apoptotic pathways. These findings could improve the understanding and enhance the development of drugs based on E. sieboldianum and raise its application value in anticancer therapy or prevention. In addition, our results indicated that Aurora kinase might be a novel target of HPDL and QGGP.

  4. Involvement of the helix-loop-helix protein Id-1 in the glucocorticoid regulation of tight junctions in mammary epithelial cells.


    Woo, P L; Cercek, A; Desprez, P Y; Firestone, G L


    Mammary epithelial cell-cell junctions undergo morphological and structural differentiation during pregnancy and lactation, but little is known about the transcriptional regulators that are involved in this process. In Con8 mammary epithelial tumor cells, we have previously documented that the synthetic glucocorticoid, dexamethasone, induces the reorganization of the tight junction and adherens junction and stimulates the monolayer transepithelial electrical resistance (TER), a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment rapidly and strongly stimulated the level of the Id-1 protein, which is a serum-inducible helix-loop-helix transcriptional repressor. The steroid induction of Id-1 was robust by 4 h of treatment and maintained over a 24-h period. Isopropyl-1-thio-beta-d-galactopyranoside-inducible expression of exogenous Id-1 in Con8 cells was shown to strongly facilitate the dexamethasone induction of TER in the absence of serum without altering the dexamethasone-dependent reorganization of ZO-1, beta-catenin, or F-actin. Ectopic overexpression of Id-1 in the SCp2 nontumorigenic mammary epithelial cells, which does not undergo complete dexamethasone-dependent tight junction reorganization, enhanced the dexamethasone-induced ZO-1 tight junction localization and stimulated the monolayer TER. Moreover, antisense reduction of Id-1 protein in SCp2 cells prevented the apical junction reorganization and dexamethasone-stimulated TER. Our results implicate Id-1 as acting as a critical regulator of mammary epithelial cell-cell interactions at an early step in the glucocorticoid-dependent signaling pathway that controls tight junction integrity.

  5. Intermittent parathyroid hormone administration converts quiescent lining cells to active osteoblasts.


    Kim, Sang Wan; Pajevic, Paola Divieti; Selig, Martin; Barry, Kevin J; Yang, Jae-Yeon; Shin, Chan Soo; Baek, Wook-Young; Kim, Jung-Eun; Kronenberg, Henry M


    Intermittent administration of parathyroid hormone (PTH) increases bone mass, at least in part, by increasing the number of osteoblasts. One possible source of osteoblasts might be conversion of inactive lining cells to osteoblasts, and indirect evidence is consistent with this hypothesis. To better understand the possible effect of PTH on lining cell activation, a lineage tracing study was conducted using an inducible gene system. Dmp1-CreERt2 mice were crossed with ROSA26R reporter mice to render targeted mature osteoblasts and their descendents, lining cells and osteocytes, detectable by 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining. Dmp1-CreERt2(+):ROSA26R mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 3, 5, 7, 14, and 21. The animals were euthanized on postnatal day 23, 33, or 43 (2, 12, or 22 days after the last 4-OHTam injection). On day 43, mice were challenged with a subcutaneous injection of human PTH (1-34, 80 µg/kg) or vehicle once daily for 3 days. By 22 days after the last 4-OHTam injection, most X-gal (+) cells on the periosteal surfaces of the calvaria and the tibia were flat. Moreover, bone formation rate and collagen I(α1) mRNA expression were decreased at day 43 compared to day 23. After 3 days of PTH injections, the thickness of X-gal (+) cells increased, as did their expression of osteocalcin and collagen I(α1) mRNA. Electron microscopy revealed X-gal-associated chromogen particles in thin cells prior to PTH administration and in cuboidal cells following PTH administration. These data support the hypothesis that intermittent PTH treatment can increase osteoblast number by converting lining cells to mature osteoblasts in vivo.

  6. Enzymatic synthesis and characterization of hydroquinone galactoside using Kluyveromyces lactis lactase.


    Kim, Go-Eun; Lee, Jin-Ha; Jung, Sun-Hwa; Seo, Eun-Seong; Jin, Sheng-De; Kim, Ghahyun J; Cha, Jaeho; Kim, Eui-Joong; Park, Ki-Deok; Kim, Doman


    Hydroquinone galactoside (HQ-Gal) as a potential skin whitening agent was synthesized by the reaction of lactase (beta-galactosidase) from Kluyveromyces lactis, Aspergillus oryzae, Bacillus circulans, and Thermus sp. with lactose as a donor and HQ as an acceptor. Among these lactases, the acceptor reaction involving HQ and lactose with K. lactis lactase showed a higher conversion ratio to HQ-Gal (60.27%). HQ-Gal was purified using butanol partitioning and silica gel column chromatography. The structure of the purified HQ-Gal was determined by nuclear magnetic resonance, and the ionic product was observed at m/z 295 (C12H16O7Na)+ using matrix assisted laser desorption ionization time-of-flight mass spectrometry. HQ-Gal was identified as 4-hydroxyphenyl-beta-d-galactopyranoside. The optimum conditions for HQ-Gal synthesis by K. lactis determined using response surface methodology were 50 mM HQ, 60 mM lactose, and 20 U mL(-1) lactase. These conditions produced a yield of 2.01 g L(-1) HQ-Gal. The half maximal inhibitory concentration (IC50) of diphenylpicrylhydrazyl scavenging activity was 3.31 mM, indicating a similar antioxidant activity compared to beta-arbutin (IC50=3.95 mM). The Ki value of HQ-Gal (0.75 mM) against tyrosinase was smaller than that of beta-arbutin (Ki=1.97 mM), indicating its superiority as an inhibitor. HQ-Gal inhibited (23%) melanin synthesis without being significantly toxic to the cells, while beta-arbutin exhibited only 8% reduction of melanin synthesis in B16 melanoma cells compared with the control. These results indicate that HQ-Gal may be a suitable functional component in the cosmetics industry.

  7. Expression of Functional Human Sialyltransferases ST3Gal1 and ST6Gal1 in Escherichia coli

    PubMed Central

    Ortiz-Soto, Maria Elena; Seibel, Jürgen


    Sialyltransferases (STs) are disulfide-containing, type II transmembrane glycoproteins that catalyze the transfer of sialic acid to proteins and lipids and participate in the synthesis of the core structure oligosaccharides of human milk. Sialic acids are found at the outermost position of glycostructures, playing a key role in health and disease. Sialylation is also essential for the production of recombinant therapeutic proteins (RTPs). Despite their importance, availability of sialyltransferases is limited due to the low levels of stable, soluble and active protein produced in bacterial expression systems, which hampers biochemical and structural studies on these enzymes and restricts biotechnological applications. We report the successful expression of active human sialyltransferases ST3Gal1 and ST6Gal1 in commercial Escherichia coli strains designed for production of disulfide-containing proteins. Fusion of hST3Gal1 with different solubility enhancers and substitution of exposed hydrophobic amino acids by negatively charged residues (supercharging-like approach) were performed to promote solubility and folding. Co-expression of sialyltransferases with the chaperon/foldases sulfhydryl oxidase, protein disulfide isomerase and disulfide isomerase C was explored to improve the formation of native disulfide bonds. Active sialyltransferases fused with maltose binding protein (MBP) were obtained in sufficient amounts for biochemical and structural studies when expressed under oxidative conditions and co-expression of folding factors increased the yields of active and properly folded sialyltransferases by 20%. Mutation of exposed hydrophobic amino acids increased recovery of active enzyme by 2.5-fold, yielding about 7 mg of purified protein per liter culture. Functionality of recombinant enzymes was evaluated in the synthesis of sialosides from the β-d-galactoside substrates lactose, N-acetyllactosamine and benzyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside

  8. Separation of four flavonol glycosides from Solanum rostratum Dunal using aqueous two-phase flotation followed by preparative high-performance liquid chromatography.


    Chang, Lin; Shao, Qian; Xi, Xingjun; Chu, Qiao; Wei, Yun


    Aqueous two-phase flotation followed by preparative high-performance liquid chromatography was used to separate four flavonol glycosides from Solanum rostratum Dunal. In the aqueous two-phase flotation section, the effects of sublation solvent, solution pH, (NH4 )2 SO4 concentration in aqueous solution, cosolvent, N2 flow rate, flotation time, and volumes of the polyethylene glycol phase on the recovery were investigated in detail, and the optimal conditions were selected: 50 wt% polyethylene glycol 1000 ethanol solvent as the flotation solvent, pH 4, 350 g/L of (NH4 )2 SO4 concentration in aqueous phase, 40 mL/min of N2 flow rate, 30 min of flotation time, 10.0 mL of flotation solvent volume, and two times. After aqueous two-phase flotation concentration, the flotation products were purified by preparative high-performance liquid chromatography. The purities of the final products A and B were 98.1 and 99.0%. Product B was the mixture of three compounds based on the analysis of high-performance liquid chromatography at the temperature of 10°C, while product A was hyperoside after the identification by nuclear magnetic resonance. Astragalin, 3'-O-methylquercetin 3-O-β-d-galactopyranoside, and 3'-O-methylquercetin 3-O-β-d-glucopyranoside were obtained with the purity of 93.8, 97.1, and 99.2%, respectively, after the further separation of product B using preparative high-performance liquid chromatography.

  9. Purification and characterization of β-glucosidase from greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae).


    Kara, Hatibe Ertürk; Turan, Yusuf; Er, Aylin; Acar, Mesut; Tümay, Sabiha; Sinan, Selma


    The greater wax moth, Galleria mellonella, is one of the most ruinous pests of honeycomb in the world. Beta-glucosidases are a type of digestive enzymes that hydrolytically catalyzes the beta-glycosidic linkage of glycosides. Characterization of the beta-glucosidase in G. mellonella could be a significant stage for a better comprehending of its role and establishing a safe and effective control procedure primarily against G. mellonella and also some other insect pests. Laboratory reared final instar stage larvae were randomly selected and homogenized for beta-glucosidase activity assay and subsequent analysis. The enzyme was purified to apparent homogeneity by salting out with ammonium sulfate and using sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purification was 58-fold with an overall enzyme yield of 29%. The molecular mass of the protein was estimated as ca. 42 kDa. The purified beta-glucosidase was effectively active on para/ortho-nitrophenyl-beta-d-glucopyranosides (p-/o-NPG) with Km values of 0.37 and 1.9 mM and Vmax values of 625 and 189 U/mg, respectively. It also exhibits different levels of activity against para-nitrophenyl-β-d-fucopyranoside (p-NPF), para/ortho-nitrophenyl β-d-galactopyranosides (p-/o-NPGal) and p-nitrophenyl 1-thio-β-d-glucopyranoside. The enzyme was competitively inhibited by beta-gluconolactone and also was very tolerant to glucose against p-NPG as substrate. The Ki and IC50 values of δ-gluconolactone were determined as 0.021 and 0.08 mM while the enzyme was more tolerant to glucose inhibition with IC50 value of 213.13 mM for p-NPG.

  10. Cloning, purification and characterization of a thermostable β-galactosidase from Bacillus licheniformis strain KG9.


    Matpan Bekler, F; Stougaard, P; Güven, K; Gül Güven, R; Acer, Ö


    A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.

  11. Screening of mango (Mangifera indica L.) cultivars for their contents of flavonol O- and xanthone C-glycosides, anthocyanins, and pectin.


    Berardini, Nicolai; Fezer, Ramona; Conrad, Jürgen; Beifuss, Uwe; Carle, Reinhold; Schieber, Andreas


    With respect to their browning potential and in consideration of a combined recovery of pectin and phenolic compounds, peels of 14 cultivars and the flesh of nine cultivars of mango (Mangifera indica L.) fruits were analyzed for their contents of flavonol O- and xanthone C-glycosides by high-performance liquid chromatography (HPLC)-diode array detection-electrospray ionization mass spectrometry (ESI-MS). While total amounts of up to 4860 mg/kg dry matter demonstrated the peels to be a rich source of phenolic compounds, only traces could be detected in the flesh. The profile of flavonol glycosides of the peels proved to be highly characteristic and may therefore serve as a tool for authenticity control of mango puree concentrate, which is often produced from unpeeled fruits and represents an important intermediate for the production of mango nectars. Two compounds were isolated by preparative HPLC, and their structures were elucidated on the basis of ESI-MS as well as NMR spectroscopy, establishing the two compounds as rhamnetin 3-O-beta-galactopyranoside and rhamnetin 3-O-beta-glucopyranoside, respectively. In the peels of red-colored cultivars, cyanidin 3-O-galactoside and an anthocyanidin hexoside so far not reported in mango could tentatively be identified. The contents and degrees of esterification of pectins extracted from the lyophilized peels ranged from 12.2 to 21.2% and from 56.3 to 65.6%, respectively, suggesting mango peels also as a promising source of high-quality pectin.

  12. Characterization and site-directed mutagenesis of an α-galactosidase from the deep-sea bacterium Bacillus megaterium.


    Xu, Haibo; Qin, Yongjun; Huang, Zongqing; Liu, Ziduo


    A novel gene (BmelA) (1323bp) encoding an α-galactosidase of 440 amino acids was cloned from the deep-sea bacterium Bacillus megaterium and the protein was expressed in Escherichia coli BL21 (DE3) with an estimated molecular mass of about 45 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 4, with the highest identity (74%) to α-galactosidase Mel4A from Bacillus halodurans among the characterized α-galactosidases. The recombinant BmelA displayed its maximum activity at 35 °C and pH 8.5-9.0 in 50 mM Tris-HCl buffer, and could hydrolyze different substrates with the Km values against p-nitrophenyl-α-D-galactopyranoside (pNP-α-Gal), raffinose and stachyose being 1.02±0.02, 2.24±0.11 and 3.42±0.17 mM, respectively. Besides, 4 mutants (I38 V, I38A, I38F and Q84A) were obtained by site-directed mutagenesis based on molecular modeling and sequence alignment. The kinetic analysis indicated that mutants I38 V and I38A exhibited a 1.7- and 1.4-fold increase over the wild type enzyme in catalytic efficiency (k(cat)/K(m)) against pNP-α-Gal, respectively, while mutant I38F showed a 3.5-fold decrease against pNP-α-Gal and mutant Q84A almost completely lost its activity. All the results suggest that I38 and Q84 sites play a vital role in enzyme activity probably due to their steric and polar effects on the predicted "tunnel" structure and NAD+ binding to the enzyme.

  13. The Structure And Synthetic Capabilities Of A Catalytic Peptide Formed By Substrate-Directed Mechanism Implications To Prebiotic Catalysis

    NASA Astrophysics Data System (ADS)

    Fleminger, Gideon; Yaron, Tal; Eisenstein, Miriam; Bar-Nun, Akiva


    Previously, we have shown that a small substrate may serve as a template in the formation of a specific catalytic peptide, a phenomenon which might have had a major role in prebiotic synthesis of peptide catalysts. This was demonstrated experimentally by the formation of a catalytic metallo-dipeptide, Cys2-Fe2+, around o-nitrophenyl β-D-galactopyranoside (ONPG), by dicyandiamide (DCDA)-assisted condensation under aqueous conditions. This dipeptide was capable of hydrolyzing ONPG at a specific activity lower only 1000 fold than that of β galactosidase. In the present paper we use molecular modeling techniques to elucidate the structure of this catalyst and its complex with the substrate and propose a putative mechanism for the catalyst formation and its mode of action as a “mini enzyme”. This model suggests that interaction of Fe2+ ion with ONPG oxygens and with two cysteine SH groups promotes the specific formation of the Cys2-Fe2+ catalyst. Similarly, the interaction of the catalyst with ONPG is mediated by its Fe2+ with the substrate oxygens, leading to its hydrolysis. In addition, immobilized forms of the catalyst were synthesized on two carriers Eupergit C and amino glass beads. These preparations were capable of catalyzing the formation of ONPG from β-D-galactose and o-nitrophenol (ONP) under anhydrous conditions. The ability of the catalyst to synthesize the substrate that mediates its own formation creates an autocatalytic cycle where ONPG catalyzes the formation of a catalyst which, in turn, catalyzes ONPG formation. Such autocatalytic cycle can only operate by switching between high and low water activity conditions, such as in tidal pools cycling between wet and dry environments. Implications of the substrate-dependent formation of catalytically active peptides to prebiotic processes are discussed

  14. The structure and synthetic capabilities of a catalytic peptide formed by substrate-directed mechanism--implications to prebiotic catalysis.


    Fleminger, Gideon; Yaron, Tal; Eisenstein, Miriam; Bar-Nun, Akiva


    Previously, we have shown that a small substrate may serve as a template in the formation of a specific catalytic peptide, a phenomenon which might have had a major role in prebiotic synthesis of peptide catalysts. This was demonstrated experimentally by the formation of a catalytic metallo-dipeptide, Cys2-Fe2+, around o-nitrophenyl beta-D-galactopyranoside (ONPG), by dicyandiamide (DCDA)-assisted condensation under aqueous conditions. This dipeptide was capable of hydrolyzing ONPG at a specific activity lower only 1000 fold than that of beta galactosidase. In the present paper we use molecular modeling techniques to elucidate the structure of this catalyst and its complex with the substrate and propose a putative mechanism for the catalyst formation and its mode of action as a "mini enzyme". This model suggests that interaction of Fe2+ ion with ONPG oxygens and with two cysteine SH groups promotes the specific formation of the Cys2-Fe2+ catalyst. Similarly, the interaction of the catalyst with ONPG is mediated by its Fe2+ with the substrate oxygens, leading to its hydrolysis. In addition, immobilized forms of the catalyst were synthesized on two carriers--Eupergit C and amino glass beads. These preparations were capable of catalyzing the formation of ONPG from beta-D-galactose and o-nitrophenol (ONP) under anhydrous conditions. The ability of the catalyst to synthesize the substrate that mediates its own formation creates an autocatalytic cycle where ONPG catalyzes the formation of a catalyst which, in turn, catalyzes ONPG formation. Such autocatalytic cycle can only operate by switching between high and low water activity conditions, such as in tidal pools cycling between wet and dry environments. Implications of the substrate-dependent formation of catalytically active peptides to prebiotic processes are discussed.

  15. Enzyme immobilization in novel alginate-chitosan core-shell microcapsules.


    Taqieddin, Ehab; Amiji, Mansoor


    Alginate-chitosan core-shell microcapsules were prepared in order to develop a biocompatible matrix for enzyme immobilization, where the protein is retained either in a liquid or solid core and the shell allows permeability control over substrates and products. The permeability coefficients of different molecular weight compounds (vitamin B2, vitamin B12, and myoglobin) were determined through sodium tripolyphosphate (Na-TPP)-crosslinked chitosan membrane. The microcapsule core was formed by crosslinking sodium alginate with either calcium or barium ions. The crosslinked alginate core was uniformly coated with a chitosan layer and crosslinked with Na-TPP. In the case of calcium alginate, the phosphate ions of Na-TPP were able to extract the calcium ions from alginate and liquefy the core. A model enzyme, beta-galactosidase, was immobilized in the alginate core and the catalytic activity was measured with o-nitrophenyl-beta-D-galactopyranoside (ONPG). Change in the activity of free and immobilized enzyme was determined at three different temperatures. Na-TPP crosslinked chitosan membranes were found to be permeable to solutes of up to 17,000Da molecular weight. The enzyme loading efficiency was higher in the barium alginate core (100%) as compared to the calcium alginate core (60%). The rate of ONPG conversion to o-nitrophenol was faster in the case of calcium alginate-chitosan microcapsules as compared to barium alginate-chitosan microcapsules. Barium alginate-chitosan microcapsules, however, did improve the stability of the enzyme at 37 degrees C relative to calcium alginate-chitosan microcapsules or free enzyme. This study illustrates a new method of enzyme immobilization for biotechnology applications using liquid or solid core and shell microcapsule technology.

  16. Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice.


    Hu, Jan C-C; Hu, Yuanyuan; Smith, Charles E; McKee, Marc D; Wright, J Timothy; Yamakoshi, Yasuo; Papagerakis, Petros; Hunter, Graeme K; Feng, Jerry Q; Yamakoshi, Fumiko; Simmer, James P


    Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam(+/-) mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam(-/-) mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam(-/-) mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization.

  17. In vivo enzyme immobilization by use of engineered polyhydroxyalkanoate synthase.


    Peters, Verena; Rehm, Bernd H A


    This study demonstrated that engineered polyhydroxyalkanoate (PHA) synthases can be employed as molecular tools to covalently immobilize enzymes at the PHA granule surface. The beta-galactosidase was fused to the N terminus of the class II PHA synthase from Pseudomonas aeruginosa. The open reading frame was confirmed to encode the complete fusion protein by T7 promoter-dependent overexpression. Restoration of PHA biosynthesis in the PHA-negative mutant of P. aeruginosa PAO1 showed a PHA synthase function of the fusion protein. PHA granules were isolated and showed beta-galactosidase activity. PHA granule attached proteins were analyzed and confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Surprisingly, the beta-galactosidase-PHA synthase fusion protein was detectable at a high copy number at the PHA granule, compared with PHA synthase alone, which was barely detectable at PHA granules. Localization of the beta-galactosidase at the PHA granule surface was confirmed by enzyme-linked immunosorbent assay using anti-beta-galactosidase antibodies. Treatment of these beta-galactosidase-PHA granules with urea suggested a covalent binding of the beta-galactosidase-PHA synthase to the PHA granule. The immobilized beta-galactosidase was enzymologically characterized, suggesting a Michaelis-Menten reaction kinetics. A Km of 630 microM and a Vmax of 17.6 nmol/min for orthonitrophenyl-beta-D-galactopyranoside as a substrate was obtained. The immobilized beta-galactosidase was stable for at least several months under various storage conditions. This study demonstrated that protein engineering of PHA synthase enables the manufacture of PHA granules with covalently attached enzymes, suggesting an application in recycling of biocatalysts, such as in fine-chemical production.

  18. Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

    PubMed Central

    Mito, S; Zhang, Q M; Yonei, S


    Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress. PMID:8386722

  19. Electroreduction-based electrochemical-enzymatic redox cycling for the detection of cancer antigen 15-3 using graphene oxide-modified indium-tin oxide electrodes.


    Park, Seonhwa; Singh, Amardeep; Kim, Sinyoung; Yang, Haesik


    We compare herein biosensing performance of two electroreduction-based electrochemical-enzymatic (EN) redox-cycling schemes [the redox cycling combined with simultaneous enzymatic amplification (one-enzyme scheme) and the redox cycling combined with preceding enzymatic amplification (two-enzyme scheme)]. To minimize unwanted side reactions in the two-enzyme scheme, β-galactosidase (Gal) and tyrosinase (Tyr) are selected as an enzyme label and a redox enzyme, respectively, and Tyr is selected as a redox enzyme label in the one-enzyme scheme. The signal amplification in the one-enzyme scheme consists of (i) enzymatic oxidation of catechol into o-benzoquinone by Tyr and (ii) electroreduction-based EN redox cycling of o-benzoquinone. The signal amplification in the two-enzyme scheme consists of (i) enzymatic conversion of phenyl β-d-galactopyranoside into phenol by Gal, (ii) enzymatic oxidation of phenol into catechol by Tyr, and (iii) electroreduction-based EN redox cycling of o-benzoquinone including further enzymatic oxidation of catechol to o-benzoquinone by Tyr. Graphene oxide-modified indium-tin oxide (GO/ITO) electrodes, simply prepared by immersing ITO electrodes in a GO-dispersed aqueous solution, are used to obtain better electrocatalytic activities toward o-benzoquinone reduction than bare ITO electrodes. The detection limits for mouse IgG, measured with GO/ITO electrodes, are lower than when measured with bare ITO electrodes. Importantly, the detection of mouse IgG using the two-enzyme scheme allows lower detection limits than that using the one-enzyme scheme, because the former gives higher signal levels at low target concentrations although the former gives lower signal levels at high concentrations. The detection limit for cancer antigen (CA) 15-3, a biomarker of breast cancer, measured using the two-enzyme scheme and GO/ITO electrodes is ca. 0.1 U/mL, indicating that the immunosensor is highly sensitive.

  20. High-level expression of Falcipain-2 in Escherichia coli by codon optimization and auto-induction.


    Sarduy, Emir Salas; Muñoz, Aymara Cabrera; Trejo, Sebastián Alejandro; de los A Chavéz Planes, María


    Falcipain-2, the major cysteine hemoglobinase from the human malaria parasite Plasmodium falciparum, is critical for parasite development and is considered a promising chemotherapeutic target. In order to facilitate the high-throughput screening of Falcipain-2 inhibitors from natural sources, we developed an economic and highly-productive overexpression system in Escherichia coli using a codon-optimized proFalcipain-2 construct. Very high expression levels (35-55% of total host proteins) were observed when proFalcaipain-2 expression was induced with 1mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) in several E. coli strains, with the highest level observed for BL21(DE3). A lower expression (~40% of total host proteins) was observed when BL21(DE3) was grown in ZYM-5052 auto-induction medium, containing 0.2% lactose as inducer. However, the culture grew to notably higher cellular density, increasing ~1.5 times the overall yield of the system when compared with conventional IPTG-induction. Although several conditions were modified to achieve the expression of soluble and active Falcipain-2, the enzyme was mainly obtained in the form of insoluble aggregates. After purification and refolding, ~50 mg of active enzyme were obtained per liter of culture at low cost using a regular incubator shaker, and recombinant Falcipain-2 exhibited structural and functional characteristics very similar to the natural counterpart. Due to its versatility and simplicity, this strategy can be straightforwardly adapted to other proteins from Plasmodium species or any other organism with an AT-rich genome.

  1. Human milk and mucosal lacto- and galacto-N-biose synthesis by transgalactosylation and their prebiotic potential in Lactobacillus species.


    Bidart, Gonzalo N; Rodríguez-Díaz, Jesús; Palomino-Schätzlein, Martina; Monedero, Vicente; Yebra, María J


    Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-D-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.

  2. Catalytic Mechanism of Human Alpha-galactosidase

    SciTech Connect

    Guce, A.; Clark, N; Salgado, E; Ivanen, D; Kulinskaya, A; Brumer, H; Garman, S


    The enzyme {alpha}-galactosidase ({alpha}-GAL, also known as {alpha}-GAL A; E.C. is responsible for the breakdown of {alpha}-galactosides in the lysosome. Defects in human {alpha}-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of {alpha}-galactosylated substrates in the tissues. {alpha}-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human {alpha}-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-{alpha}-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a {sup 1}S{sub 3} skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on {alpha}-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

  3. Purification and chemical characterisation of a cell wall-associated β-galactosidase from mature sweet cherry (Prunus avium L.) fruit.


    Gerardi, Carmela; Blando, Federica; Santino, Angelo


    Using four different chromatographic steps, β-galactosidase was purified from the ripe fruit of sweet cherry to apparent electrophoretic homogeneity with approximately 131-fold purification. The Prunus avium β-galactosidase showed an apparent molecular mass of about 100 kDa and consisted of four different active polypeptides with pIs of about 7.9, 7.4, 6.9 and 6.4 as estimated by native IEF and β-galactosidase-activity staining. The active polypeptides were individually excised from the gel and subjected to SDS-PAGE. Each of the four native enzymes showing β-galactosidase activity was composed of two polypeptides with an estimated mass of 54 and 33 kDa. Both of these polypeptides were subjected to N-terminal amino acid sequence analysis. The 54 kDa polypeptide of sweet cherry β-galactosidase showed a 43% identity with the 44 kDa subunit of persimmon and apple β-galactosidases and the 48 kDa subunit of carambola galactosidase I. The sweet cherry β-galactosidase exhibited a strict specificity towards p-nitrophenyl β-D-galactopyranoside, a pH optimum of 4.0 and K(m) and V(max) values of 0.42 mM and 4.12 mmol min(-1) mg(-1) of protein respectively with this substrate. The enzyme was also active towards complex glycans. Taken together the results of this study prompted a role for this class of enzymes on sweet cherry fruit ripening and softening.

  4. Cyclooxygenase inhibitory and antioxidant compounds from crabapple fruits.


    Seeram, Navindra P; Cichewicz, Robert H; Chandra, Amitabh; Nair, Muraleedharan G


    Crabapple trees belong to the Malus genus (Rosaceae) and bear fruits that are sparingly consumed and used in the preparation of fruit beverages. Cyclooxygenase (COX) enzyme inhibitory and antioxidant bioassay-guided fractionation of the aqueous and methanol extracts of Malus x kornicensis and Malus x Indian Summer yielded (+)-catechin (1), (-)-epicatechin (2), cyanidin-3-O-beta-galactopyranoside (3), and amygdalin (4). Pure compounds 1-4 were obtained by HPLC, identified by LC-ES/MS, CD, and NMR spectroscopic methods and evaluated for their COX enzyme inhibitory and antioxidant activities. In COX-1 and -2 enzyme inhibitory assays, compounds 1-3 (all at 80 microM) showed activities of 20.4, 46.3%; 57.6, 47.9%; and 8.2, 13.7%, respectively, compared to naproxen (54.3, 41.3%; 10 microM), ibuprofen (47.5, 39.8%; 10 microM), Celebrex (46.2, 66.3%; 1.67 ppm), and Vioxx (23.8, 88.1%, 1.67 ppm). In the antioxidant assay, the catechins (1-2) and anthocyanin (3) (all at 40 microM) showed activities of 61.3, 62.5, and 60.1%, respectively. The synthetic antioxidants, tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and vitamin E (all tested at 10 microM), gave 75.2, 80.1, 70.0, and 10.2% activities, respectively. The cyanogenic glycoside, amygdalin (4), and its hydrolysis products, mandelonitrile (5) and benzaldehyde (6), were not active in the antioxidant or COX enzyme inhibitory assays at 80 microM concentrations.

  5. Presence, fate and effects of the intense sweetener sucralose in the aquatic environment.


    Tollefsen, Knut Erik; Nizzetto, Luca; Huggett, Duane B


    Sucralose (1,6-dichloro-1,6-dideoxy-b-D-fructo-furanosyl 4-chloro-4-deoxy-a-D-galactopyranoside), sold under the trade name Splenda, has been detected in municipal effluents and surface waters in the United States and Europe. The environmental presence of sucralose has led to interest in the possibility of toxic effects in non-target species. This review presents an environmental risk assessment of sucralose based on available data concerning its presence, fate and effects in the environment. Sucralose, which is made by selective chlorination of sucrose, is a highly stable compound, which undergoes negligible metabolism in mammals, including humans, and displays a low biodegradation potential in the environment. This intense sweetener is highly soluble in water, displays a low bioaccumulation potential and a low sorption potential to soil and organic matter, and thus is predominantly present in the water column. The predicted environmental concentration (PEC) for sucralose, based on measured data in surface waters, was determined to be 10 μg/L. Aquatic toxicity studies using standardized, validated protocols used in regulatory decision making indicate that sucralose does not alter survival, growth and reproduction of aquatic organisms (such as plants, algae, crustaceans and fish) at concentrations >9000 times higher than those detected in the environment. Some studies, using non-standardized protocols, have reported behavioral and other non-traditional responses in aquatic organisms, but the relevance of these findings for assessing adverse effects on individuals and populations will require further investigation. In terms of traditional risk assessment, the proposed predicted no effect concentration for aquatic organisms (PNEC) was determined to be 0.93 mg/L, based on the lowest no effect concentration (NOEC) from a validated chronic study with mysid shrimp and an application factor of 100. The resultant PEC/PNEC quotient was determined to be well below 1 (PEC

  6. Identification and Biochemical Characterization of Sco3487 from Streptomyces coelicolor A3(2), an Exo- and Endo-Type β-Agarase-Producing Neoagarobiose

    PubMed Central

    Temuujin, Uyangaa; Chi, Won-Jae; Chang, Yong-Keun


    Streptomyces coelicolor can degrade agar, the main cell wall component of red macroalgae, for growth. To constitute a crucial carbon source for bacterial growth, the alternating α-(1,3) and β-(1,4) linkages between the 3,6-anhydro-l-galactoses and d-galactoses of agar must be hydrolyzed by α/β-agarases. In S. coelicolor, DagA was confirmed to be an endo-type β-agarase that degrades agar into neoagarotetraose and neoagarohexaose. Genomic sequencing data of S. coelicolor revealed that Sco3487, annotated as a putative hydrolase, has high similarity to the glycoside hydrolase (GH) GH50 β-agarases. Sco3487 encodes a primary translation product (88.5 kDa) of 798 amino acids, including a 45-amino-acid signal peptide. The sco3487 gene was cloned and expressed under the control of the ermE promoter in Streptomyces lividans TK24. β-Agarase activity was detected in transformant culture broth using the artificial chromogenic substrate p-nitrophenyl-β-d-galactopyranoside. Mature Sco3487 (83.9 kDa) was purified 52-fold with a yield of 66% from the culture broth. The optimum pH and temperature for Sco3487 activity were 7.0 and 40°C, respectively. The Km and Vmax for agarose were 4.87 mg/ml (4 × 10−5 M) and 10.75 U/mg, respectively. Sco3487 did not require metal ions for its activity, but severe inhibition by Mn2+ and Cu2+ was observed. Thin-layer chromatography analysis, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and Fourier transform-nuclear magnetic resonance spectrometry of the Sco3487 hydrolysis products revealed that Sco3487 is both an exo- and endo-type β-agarase that degrades agarose, neoagarotetraose, and neoagarohexaose into neoagarobiose. PMID:22020647

  7. Globoid cell leukodystrophy (Krabbe disease): normal umbilical cord blood galactocerebrosidase activity and polymorphic mutations.


    Raghavan, S; Zeng, B; Torres, P A; Pastores, G M; Kolodny, E H; Kurtzberg, J; Krivit, W


    Globoid cell leukodystrophy is an inherited metabolic disorder of the central nervous system caused by deficiency of the lysosomal enzyme galactocerebrosidase. Haematopoietic stem cell transplantation is the only available effective treatment. The engraftment from normal donors provides competent cells able to correct the metabolic defect. Umbilical cord blood cells have proved to significantly decrease complications and improve engraftment rate compared to adult marrow cells in haematopoietic stem cell transplantation. Umbilical cord blood cells must be of sufficient activity to provide central nervous system recovery after engraftment is obtained. Galactocerebrosidase activity is known to be affected by two polymorphic alleles found at nucleotides 502 and 1637 of the cDNA for this gene. This enzyme activity and the polymorphic alleles noted above were analysed in 83 random samples of umbilical cord blood. The activity, assayed with the fluorogenic substrate 6-hexadecanoylamino-4-methylumbelliferyl-beta-galactopyranoside, in those with neither polymorphic allele was 4.6 +/- 1.7 units (nmol/h per mg protein). This optimal choice of cord blood was found in only 24% of specimens. Homozygotes for 1637T > C with activity of only 1.5 +/- 0.4 units represented 16% of the samples. Those heterozygous for 1637T > C with slightly better activity (2.3 +/- 0.7 units) represented 52% of the samples. Choice of umbilical cord blood for haematopoietic stem cell transplantation, therefore, requires consideration not only of cell quantity and HLA compatibility but also selection for normal alleles to obtain maximal enzymatic activity for central nervous system correction.

  8. A novel agar formulation for isolation and direct enumeration of Vibrio vulnificus from oyster tissue.


    Griffitt, Kimberly J; Grimes, D Jay


    A new selective and differential medium, Vibrio vulnificus X-Gal (VVX), was developed for direct enumeration of V. vulnificus (Vv) from oyster samples. This agar utilizes cellobiose and lactose as carbon sources, and the antibiotics colistin and polymyxin B as selective agents. Hydrolysis of 5-bromo-4-chloro-3-indolyl- beta-d-galactopyranoside (x-gal), used in the agar as a lactose analog, produces an insoluble blue dye that makes lactose positive colonies easily distinguishable from any non-lactose fermenting bacteria. Various bacterial species were spot plated onto thiosulfate-citrate-bile salts-sucrose agar (TCBS), and CHROMagar Vibrio, two vibrio-specific selective agars, non-selective agar, and VVX to compare selectivity of VVX to other widely used media. A V. vulnificus pure culture was serially diluted on VVX and non-selective agar to determine the VVX percent recovery. Water and oyster samples were spread plated on VVX agar and allowed to incubate for 16-18 h at 33 °C. Blue and white colonies from VVX agar were picked and screened by end point PCR for the Vv hemolysin vvhA. VVX agar showed a significant improvement over TCBS and CHROMagar at preventing non-target growth. There was an 87.5% recovery compared to non-selective plating and a 98% positivity rate of blue colonies picked from oyster tissue plating. The findings suggest that this new agar is a fast, distinctive, and accurate method for enumeration of V. vulnificus from the environment.

  9. A highly parallel microfluidic droplet method enabling single-molecule counting for digital enzyme detection

    PubMed Central

    Guan, Zhichao; Zou, Yuan; Zhang, Mingxia; Lv, Jiangquan; Shen, Huali; Yang, Pengyuan; Zhang, Huimin; Zhu, Zhi; James Yang, Chaoyong


    Although digital detection of nucleic acids has been achieved by amplification of single templates in uniform microfluidic droplets and widely used for genetic analysis, droplet-based digital detection of proteins has rarely been reported, largely due to the lack of an efficient target amplification method for protein in droplets. Here, we report a key step towards digital detection of proteins using a highly parallel microfluidic droplet approach for single enzyme molecule detection in picoliter droplets via enzyme catalyzed signal amplification. An integrated microfluidic chip was designed for high throughput uniform droplet generation, monolayer droplet collection, incubation, detection, and release. Single β-galatosidase (β-Gal) molecules and the fluorogenic substrate fluorescein di-β-D-galactopyranoside were injected from two separated inlets to form uniform 20 μm droplets in fluorinated oil at a frequency of 6.6 kHz. About 200 000 droplets were captured as a monolayer in a capture well on-chip for subsequent imaging detection. A series of β-Gal solutions at different concentrations were analyzed at the single-molecule level. With no enzyme present, no droplets were found to fluoresce, while brightly fluorescent droplets were observed under single-enzyme molecule conditions. Droplet fluorescence intensity distribution analysis showed that the distribution of enzyme molecules under single-molecule conditions matched well with theoretical prediction, further proving the feasibility of detecting single enzyme molecules in emulsion droplets. Moreover, the population of fluorescent droplets increased as the β-Gal concentration increased. Based on a digital counting method, the measured concentrations of the enzyme were found to match well with input enzyme concentration, establishing the accuracy of the digital detection method for the quantification of β-Gal enzyme molecules. The capability of highly parallel detection of single enzyme molecules in

  10. An interference-free and rapid electrochemical lateral-flow immunoassay for one-step ultrasensitive detection with serum.


    Akanda, Md Rajibul; Joung, Hyou-Arm; Tamilavan, Vellaiappillai; Park, Seonhwa; Kim, Sinyoung; Hyun, Myung Ho; Kim, Min-Gon; Yang, Haesik


    Point-of-care testing (POCT) of biomarkers in clinical samples is of great importance for rapid and cost-effective diagnosis. However, it is extremely challenging to develop an electrochemical POCT technique retaining both ultrasensitivity and simplicity. We report an interference-free electrochemical lateral-flow immunoassay that enables one-step ultrasensitive detection with serum. The electrochemical-chemical-chemical (ECC) redox cycling combined with an enzymatic reaction of an enzyme label is used to obtain high signal amplification. The ECC redox cycling involving Ru(NH3)6(3+), enzyme product, and tris(3-carboxyethyl)phosphine (TCEP) depends on pH, because the formal potentials of an enzyme product and TCEP increase with decreasing pH although that of Ru(NH3)6(3+) is pH-independent. With consideration of the pH dependence of ECC redox cycling, a noble combination of enzyme label, substrate, and product [β-galactosidase, 4-amino-1-naphthyl β-D-galactopyranoside, and 4-amino-1-naphthol, respectively] is introduced to ensure fast and selective ECC redox cycling of the enzyme product along with a low background level. The selective ECC redox cycling at a low applied potential (0.05 V vs. Ag/AgCl) minimizes the interference effect of electroactive species (L-ascorbic acid, acetaminophen, and uric acid) in serum. A detection limit of 0.1 pg mL(-1) for troponin I is obtained only 11 min after serum dropping without the use of an additional solution. Moreover, the lateral-flow immunoassay is applicable to the analysis of real clinical samples.

  11. Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.


    Militello, Kevin T; Chang, Ming-Mei; Simon, Robert D; Lazatin, Justine C


    The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl β-D-thiogalactopyranoside (IPTG). Over the course of the next three weeks, students use a battery of approaches including plasmid DNA isolation experiments, restriction maps, and PCR to differentiate between mutation and transformation. The students ultimately come to the conclusion that the changes in phenotypes are due to genetic transformation and not mutation based on the evidence generated over the four-week period. Pre-laboratory tests and post-laboratory tests indicate that this set of exercises is successful in helping students differentiate between transformation and mutation. The laboratory is designed for underclassmen and is a good prerequisite for an apprentice-based research opportunity, although it is not designed as a class based research experience. Potential modifications and future directions of the laboratory based upon student experiences and assessment are presented.

  12. Kinetic Analysis of Guanidine Hydrochloride Inactivation of β-Galactosidase in the Presence of Galactose

    PubMed Central

    Nwamba, Charles O.; Chilaka, Ferdinand C.


    Inactivation of purified β-Galactosidase was done with GdnHCl in the absence and presence of varying [galactose] at 50°C and at pH 4.5. Lineweaver-Burk plots of initial velocity data, in the presence and absence of guanidine hydrochloride (GdnHCl) and galactose, were used to determine the relevant Km and Vmax values, with p-nitrophenyl β-D-galactopyranoside (pNPG) as substrate, S. Plots of ln([P]∞ − [P]t) against time in the presence of GdnHCl yielded the inactivation rate constant, A. Plots of A versus [S] at different galactose concentrations were straight lines that became increasingly less steep as the [galactose] increased, showing that A was dependent on [S]. Slopes and intercepts of the 1/[P]∞ versus 1/[S] yielded k+0 and k'+0, the microscopic rate constants for the free enzyme and the enzyme-substrate complex, respectively. Plots of k+0 and k'+0 versus [galactose] showed that galactose protected the free enzyme as well as the enzyme-substrate complex (only at the lowest and highest [galactose]) against GdnHCl inactivation. In the absence of galactose, GdnHCl exhibited some degree of non-competitive inhibition. In the presence of GdnHCl, galactose exhibited competitive inhibition at the lower [galactose] of 5 mM which changed to non-competitive as the [galactose] increased. The implications of our findings are further discussed. PMID:23008759

  13. Phytochemicals of apple peels: isolation, structure elucidation, and their antiproliferative and antioxidant activities.


    He, Xiangjiu; Liu, Rui Hai


    Bioactivity-guided fractionation of Red Delicious apple peels was used to determine the chemical identity of bioactive constituents, which showed potent antiproliferative and antioxidant activities. Twenty-nine compounds, including triterpenoids, flavonoids, organic acids and plant sterols, were isolated using gradient solvent fractionation, Diaion HP-20, silica gel, and ODS columns, and preparative HPLC. Their chemical structures were identified using HR-MS and 1D and 2D NMR. Antiproliferative activities of isolated pure compounds against HepG2 human liver cancer cells and MCF-7 human breast cancer cells were evaluated. On the basis of the yields of isolated flavonoids (compounds 18- 23), the major flavonoids in apple peels are quercetin-3-O-beta-D-glucopyranoside (compound 20, 82.6%), then quercetin-3-O-beta-D-galactopyranoside (compound 19, 17.1%), followed by trace amounts of quercetin (compound 18, 0.2%), (-)-catechin (compound 22), (-)-epicatechin (compound 23), and quercetin-3-O-alpha-L-arabinofuranoside (compound 21). Among the compounds isolated, quercetin (18) and quercetin-3-O-beta-D-glucopyranoside (20) showed potent antiproliferative activities against HepG2 and MCF-7 cells, with EC 50 values of 40.9 +/- 1.1 and 49.2 +/- 4.9 microM to HepG2 cells and 137.5 +/- 2.6 and 23.9 +/- 3.9 microM to MCF-7 cells, respectively. Six flavonoids (18-23) and three phenolic compounds (10, 11, and 14) showed potent antioxidant activities. Caffeic acid (10), quercetin (18), and quercetin-3-O-beta-D-arabinofuranoside (21) showed higher antioxidant activity, with EC 50 values of <10 microM. Most tested flavonoids and phenolic compounds had high antioxidant activity when compared to ascorbic acid and might be responsible for the antioxidant activities of apples. These results showed apple peel phytochemicals have potent antioxidant and antiproliferative activities.

  14. Anti-diabetic properties of flavonoid compounds isolated from Hyphaene thebaica epicarp on alloxan induced diabetic rats

    PubMed Central

    Salib, Josline Y.; Michael, Helana N.; Eskande, Emad Fawzy


    Background: Diabetes mellitus, becoming the third killer of mankind after cancer and cardiovascular diseases, is one of the most challenging diseases facing health care professionals today. That is why; there has been a growing interest in the therapeutic use of natural products for diabetes, especially those derived from plants. Aim: To evaluate the anti-diabetic activity together with the accompanying biological effects of the fractions and the new natural compounds of Hyphaene thebaica (HT) epicarp. Materials and Methods: 500 g of coarsely powdered of (HT) fruits epicarp were extracted by acetone. The acetone crude extract was fractionated with methanol and ethyl acetate leaving a residual water-soluble fraction WF. The anti-diabetic effects of the WF and one of its compounds of the acetone extract of the (HT) epicarp were investigated in this study using 40 adult male rats. Results: Phytochemical investigation of active WF revealed the presence of ten different flavonoids, among which two new natural compounds luteolin 7-O-[6”-O-α-Lrhamnopyranosyl]-β-D-galactopyranoside 3 and chrysoeriol 7-O-β-D-galactopyranosyl(1→2)-α-L-arabinofuranoside 5 were isolated. Supplementation of the WF improved glucose and insulin tolerance and significantly lowered blood glycosylated hemoglobin levels. On the other hand, compound 5 significantly reduced AST and ALT levels of liver, respectively. Likewise, the kidney functions were improved for both WF and compound 5, whereby both urea and creatinine levels in serum were highly significant Conclusion: The results justify the use of WF and compound 5 of the (HT) epicarp as anti-diabetic agent, taking into consideration that the contents of WF were mainly flavonoids PMID:23598921

  15. Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications

    PubMed Central

    Rico-Díaz, Agustín; Álvarez-Cao, María-Efigenia; Escuder-Rodríguez, Juan-José; González-Siso, María-Isabel; Cerdán, M. Esperanza; Becerra, Manuel


    Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45 °C compared to Kl-β-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5 °C, respectively, higher than Kl-β-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-β-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-β-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-β-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis. PMID:28361909

  16. Clinically applicable procedure for gene delivery to fetal gut by ultrasound-guided gastric injection: toward prenatal prevention of early-onset intestinal diseases.


    David, A L; Peebles, D M; Gregory, L; Waddington, S N; Themis, M; Weisz, B; Ruthe, A; Lawrence, L; Cook, T; Rodeck, C H; Coutelle, C


    Targeting gene therapy vectors to the fetal intestinal tract could provide a novel means toward prevention of the early postnatal intestinal pathology of cystic fibrosis and other conditions, such as congenital enteropathy, that cause intestinal failure. Among these conditions, cystic fibrosis is by far the most common lethal genetic disease. It is caused by a functional absence or deficiency of the cystic fibrosis transmembrane conductance regulator and manifests in the gut as meconium ileus. Prenatal treatment of genetic disease may avoid early-onset tissue damage and immune sensitization, and may target cells that are less accessible in the adult. We investigated gene transfer to the fetal gut, using a minimally invasive injection technique. First-generation replication-deficient adenoviral vectors encoding the beta-galactosidase gene and transduction-enhancing agents were injected into the stomach of early-gestation fetal sheep (n = 8, 60 days of gestation; term, 145 days) under ultrasound guidance. Reporter gene expression was observed 2 days after injection in the villi of the gastrointestinal epithelia after 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining and beta-galactosidase immunohistochemistry of fetal tissues. Expression of beta-galactosidase, as measured by enzyme-linked immunosorbent assay, was enhanced after pretreatment of the fetal gut with sodium caprate, which opens tight junctions, and after adenovirus complexation with DEAE-dextran, which confers a positive charge to the virus. Instillation of the fluorocarbon perflubron after virus delivery resulted in tissue transduction from the fetal stomach to the colon. Using a clinically relevant technique, we have demonstrated widespread gene transfer to the fetal gastrointestinal epithelia.

  17. Systemic therapy of spontaneous prostate cancer in transgenic mice with oncolytic herpes simplex viruses.


    Varghese, Susan; Rabkin, Samuel D; Nielsen, G Petur; MacGarvey, Usha; Liu, Renbin; Martuza, Robert L


    Oncolytic viruses are an innovative therapeutic strategy for cancer, wherein viral replication and cytotoxicity are selective for tumor cells. Here we show the efficacy of systemically administered oncolytic viruses for the treatment of spontaneously arising tumors, specifically the use of oncolytic herpes simplex viruses (HSV) administered i.v. to treat spontaneously developing primary and metastatic prostate cancer in the transgenic TRAMP mouse, which recapitulates human prostate cancer progression. Four administrations of systemically delivered NV1023 virus, an HSV-1/HSV-2 oncolytic recombinant, to TRAMP mice at 12 or 18 weeks of age (presence of prostate adenocarcinoma or metastatic disease, respectively) inhibited primary tumor growth and metastases to lymph nodes. Expression of interleukin 12 (IL-12) from NV1042 virus, a derivative of NV1023, was additionally effective, significantly reducing the frequency of development of prostate cancer and lung metastases, even when the mice were treated after the onset of metastasis at 18 weeks of age. NV1042-infected cells, as detected by 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside staining for Lac Z expressed by the virus, were present in prostate tumors 1 week after the final virus injection and viral DNA was detected at 2 weeks after final virus injection by real-time PCR in primary and metastatic tumors but not in liver or blood. No toxicity was observed in any of the treated mice. The efficacy of the IL-12-expressing NV1042 virus in this aggressive prostate cancer model using a clinically relevant treatment paradigm merits its consideration for clinical studies.

  18. Molecular characterization of Salmonella spp. isolated from bulk tank milk and cull dairy cow fecal samples.


    Murinda, S E; Nguyen, L T; Ivey, S J; Gillespie, B E; Almeida, R A; Draughon, F A; Oliver, S P


    The consumption of meat from cull dairy cows and of raw milk has been associated with foodborne salmonellosis. This survey was conducted to establish the prevalence of Salmonella in cull dairy cow fecal samples and bulk tank milk and to determine the proportion of Salmonella-positive dairy farms (n = 30) in east Tennessee. Food and Drug Administration bacteriological analytical protocols were generally used for Salmonella isolation. Primary enrichment was performed with lactose broth, and secondary enrichment was conducted with tetrathionate broth. Eosin methylene blue, hektoen enteric, xylose lysine desoxycholate, bismuth sulfite, and brilliant green (BG) were used as isolation agars. BG agars supplemented with individual antibiotics and/or sulfur compounds were also evaluated. Six of 268 (2.24%) bulk tank milk samples and 9 of 415 (2.17%) fecal samples from 7 of 30 (25.3%) dairy farms were Salmonella-positive. Most isolates (11 of 15) were obtained between September and December. Salmonella isolates were further characterized using polyvalent somatic O Salmonella antiserum, o-nitrophenyl-beta-D-galactopyranoside (ONPG), and Analytical Profile Index (API) 20E strips for Enterobacteriaceae. Serological evaluation of presumptive positive Salmonella isolates resulted in substantial numbers of false positives (41.2%). ONPG and API 20E tests enabled further biochemical distinction of the majority of Salmonella spp. from Salmonella Arizonae and closely related members of Enterobacteriaceae like Citrobacter youngae. Pulsed-field gel electrophoresis of SpeI-digested Salmonella DNA was used to subtype isolates. The isolates grouped into four clusters. The baseline information generated in this survey is being used to develop preharvest pathogen reduction programs on selected farms.

  19. In vivo gene delivery into ocular tissues by eye drops of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles.


    Liaw, J; Chang, S F; Hsiao, F C


    The primary objective of this study was to investigate the feasibility of using PEO-PPO-PEO non-ionic copolymeric micelles as a carrier for eye-drop gene delivery of plasmid DNA with lacZ gene in vivo. Using pyrene fluorescence probe methods, zeta potential, and dynamic light scattering test (DLS), the ability of micelle formation of these block copolymers with plasmid was studied. Gene expressions were visualized by both the quality of enzymatic color reaction using X-gal staining and by the quantification of the substrate chlorophenol red galactopyranoside (CPRG) in enucleated eyes on day 2 after gene transfer. In addition, microscopy to identify the types of cell showing uptake and expression of the transferred gene was used. We found that the block polymeric micelles were formed above 0.1% (w/v) of block copolymer with a size of 160 nm and a zeta potential of -4.4 mV. After 2 days of topically delivery three times a day, the most intense gene expression was observed on days 2 and 3. Reporter expression was detected around the iris, sclera, conjunctiva, and lateral rectus muscle of rabbit eyes and also in the intraocular tissues of nude mice upon in vivo topical application for 48 h with a DNA/polymeric micelle formulation. Furthermore, after two enhancement treatments, the transport mechanisms of the block copolymeric micelles were found through endocytosis in tissues by enhancement through the tight junction pathway. Thus, efficient and stable transfer of the functional gene could be achieved with PEO-PPO-PEO polymeric micelles through topical delivery in mice and rabbits. These in vivo experiments indicate the possible potential use of block copolymers for DNA transfer.

  20. Identification of the transcriptional regulatory sequences of human calponin promoter and their use in targeting a conditionally replicating herpes vector to malignant human soft tissue and bone tumors.


    Yamamura, H; Hashio, M; Noguchi, M; Sugenoya, Y; Osakada, M; Hirano, N; Sasaki, Y; Yoden, T; Awata, N; Araki, N; Tatsuta, M; Miyatake, S I; Takahashi, K


    The calponin (basic or h1) gene, normally expressed in maturated smooth muscle cells, is aberrantly expressed in a variety of human soft tissue and bone tumors. In this study, we show that expression of the calponin gene in human soft tissue and bone tumor cells is regulated at the transcriptional level by the sequence between positions -260 and -219 upstream of the translation initiation site. A novel conditionally replicating herpes simplex virus-1 vector (d12.CALP) in which the calponin promoter drives expression of ICP4, a major trans-activating factor for viral genes was constructed and tested as an experimental treatment for malignant human soft tissue and bone tumors. In cell culture, d12.CALP at low multiplicity of infection (0.001 plaque-forming unit/cell) selectively killed calponin-positive human synovial sarcoma, leiomyosarcoma, and osteosarcoma cells. For in vivo studies, 10 animals harboring SK-LMS-1 human leiomyosarcoma cells were randomly divided and treated twice on days 0 and 9 intraneoplastically with either 1 x 10(7) plaque-forming units of d12.CALP/100 mm(3) of tumor volume or with medium alone. The viral treatment group showed stable and significant inhibition of tumorigenicity with apparent cure in four of five mice by day 35. Replication of viral DNA demonstrated by PCR amplification and expression of the inserted LacZ gene visualized by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside histochemistry was associated with oncolysis of d12.CALP-treated tumors, while sparing normal vascular smooth muscle cells. In mice harboring two SK-LMS-1 tumors, replication of d12.CALP was detected in a nontreated tumor distant from the site of virus inoculation. These results indicate that replication-competent virus vectors controlled by the calponin transcriptional regulatory sequence may be a new therapeutic strategy for treatment of malignant human soft tissue and bone tumors.

  1. The influence of surface carbohydrates during in vitro infection of mammalian cells by the dermatophyte Trichophyton rubrum.


    Esquenazi, Daniele; Alviano, Celuta S; de Souza, Wanderley; Rozental, Sonia


    In order to better understand the role played by surface glycoconjugates during host cell adhesion and endocytosis of Trichophyton rubrum, we looked for the presence of carbohydrate-binding adhesins on the microconidia surface and their role on cellular interaction with epithelial and macrophages cells. The interaction of T. rubrum with chinese hamster ovary epithelial cells and their glycosylation-deficient mutants demonstrated a higher adhesion index in Lec1 and Lec2 mutants, that express mannose and galactose, respectively. Endocytosed fungi were shown preferentially in Lec2 cells. Addition of the carbohydrates to the interaction medium, pretreatment with lectins and with sodium periodate decreased the adhesion and endocytic index for all mutants. The ability of the fungus to penetrate into mammalian cells was confirmed in experiments using macrophages treated with cytochalasin D. Flow cytometric analysis showed that this fungus recognizes mannose and galactose. The binding was inhibited by the addition of methyl alpha-D-mannopyranoside and methyl alpha-D-galactopyranoside, and showed higher fluorescence intensity at 37 than at 28 degrees C. Trypsin treatment and heating of the cells reduced the binding, suggesting a (glyco) protein nature for the microconidia adhesins. The presence of lectin-like molecules in fungus cell could be observed by scanning electron microscopy of the fungus incubated with colloidal-gold labeled neoglycoproteins. Our results suggest that T. rubrum has the ability to invade mammalian cells and expresses carbohydrate-specific adhesins on microconidia surface that recognize mannose and galactose. These adhesins may play an important role on the adhesion and invasion of the fungus during the infectious process of dermatophytosis.

  2. Multiplexed paper test strip for quantitative bacterial detection.


    Hossain, S M Zakir; Ozimok, Cory; Sicard, Clémence; Aguirre, Sergio D; Ali, M Monsur; Li, Yingfu; Brennan, John D


    Rapid, sensitive, on-site detection of bacteria without a need for sophisticated equipment or skilled personnel is extremely important in clinical settings and rapid response scenarios, as well as in resource-limited settings. Here, we report a novel approach for selective and ultra-sensitive multiplexed detection of Escherichia coli (non-pathogenic or pathogenic) using a lab-on-paper test strip (bioactive paper) based on intracellular enzyme (β-galactosidase (B-GAL) or β-glucuronidase (GUS)) activity. The test strip is composed of a paper support (0.5 × 8 cm), onto which either 5-bromo-4-chloro-3-indolyl-β-D: -glucuronide sodium salt (XG), chlorophenol red β-galactopyranoside (CPRG) or both and FeCl(3) were entrapped using sol-gel-derived silica inks in different zones via an ink-jet printing technique. The sample was lysed and assayed via lateral flow through the FeCl(3) zone to the substrate area to initiate rapid enzyme hydrolysis of the substrate, causing a change from colorless-to-blue (XG hydrolyzed by GUS, indication of nonpathogenic E. coli) and/or yellow to red-magenta (CPRG hydrolyzed by B-GAL, indication of total coliforms). Using immunomagnetic nanoparticles for selective preconcentration, the limit of detection was ~5 colony-forming units (cfu) per milliliter for E. coli O157:H7 and ~20 cfu/mL for E. coli BL21, within 30 min without cell culturing. Thus, these paper test strips could be suitable for detection of viable total coliforms and pathogens in bathing water samples. Moreover, inclusion of a culturing step allows detection of less than 1 cfu in 100 mL within 8 h, making the paper tests strips relevant for detection of multiple pathogens and total coliform bacteria in beverage and food samples.

  3. Vibrio cincinnatiensis sp. nov., a new human pathogen.

    PubMed Central

    Brayton, P R; Bode, R B; Colwell, R R; MacDonell, M T; Hall, H L; Grimes, D J; West, P A; Bryant, T N


    A halophilic gram-negative rod was isolated from blood and cerebrospinal fluid collected from a 70-year-old male having no known contact with seafood or salt water. Positive biochemical tests included oxidase, sensitivity to 0/129, O-nitrophenyl-beta-D-galactopyranoside, lysine decarboxylase and fermentation of glucose, salicin, n-inositol, sucrose, L-mannose, L-arabinose, and arbutin. Negative tests included indole, ornithine decarboxylase, arginine dihydrolase fermentation of lactose, and production of gelatinase and urease. The DNA base composition was 45.0 mol% guanine plus cytosine. Numerical taxonomy indicated 70% similarity with known reference Vibrio sp. strains. The 5S rRNA sequence for this strain has been determined: 5'-U G C C U G G C G A C C A U A G C G U U U U G G A C C C A C C U G A U U C C A U G C C G A A C U C A G U A G U G A A A C G A A A C A G C G U C G A U G G U A G U G U G G G G U C U C C C C A U G U G A G A G U A G A A C A U C G C C A G G C A U-3'. Based on the phenetic, molecular genetic, and nucleic acid sequencing data, it is concluded that Vibrio cincinnatiensis represents a new species of the genus Vibrio sensu strictu (as defined by 5S rRNA sequencing results). On a basis of 5S rRNA comparative sequence analysis, the organism appears to share a recent common ancestor with V. gazogenes (98% homology) and close ancestry with V. mimicus, V. fluvialis, and V. metschnikovii. PMID:2422196

  4. Anti-inflammatory and free radial scavenging activities of the constituents isolated from Machilus zuihoensis.


    Mao, Yi-Wen; Tseng, Hsiang-Wen; Liang, Wen-Li; Chen, Ih-Sheng; Chen, Shui-Tein; Lee, Mei-Hsien


    A new biflavonol glycoside, quercetin-3-O-β-D-glucopyranoside-(3'→O-3''')-quercetin-3-O-β-D-galactopyranoside (9), together with eight known compounds was isolated for the first time from the leaves of Machilus zuihoensis Hayata (Lauraceae). The structure of compound 9 was elucidated by various types of spectroscopic data analysis. Analysis of the biological activity assay found that compound 9 showed significant superoxide anion scavenging activity (IC₅₀ is 30.4 μM) and markedly suppressed LPS-induced high mobility group box 1 (HMGB-1) protein secretion in RAW264.7 cells. In addition, the HMGB-1 protein secretion was also inhibited by quercitrin (3), ethyl caffeate (6), and ethyl 3-O-caffeoylquinate (7) treatment. In the LPS-stimulated inducible nitric oxide synthase (iNOS) activation analysis, two known compounds, quercetin (1) and ethyl caffeate (6), were found to markedly suppress nitric oxide (NO) production (IC₅₀ value, 27.6 and 42.9 μM, respectively) in RAW264.7 cells. Additionally, it was determined that ethyl caffeate (6) down-regulated mRNA expressions of iNOS, IL-1β, and IL-10 in the LPS-treatment of RAW264.7 cells via a suppressed NF-kB pathway. These results suggested for the first time that the new compound 9 and other constituents isolated from M. zuihoensis have potential anti-inflammatory and superoxide anion scavenging effects. These constituents may be useful for treating various inflammatory diseases.

  5. Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae.


    Tavoulari, Sotiria; Frillingos, Stathis


    We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-beta,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.

  6. The effects of organic solvents on the efficiency and regioselectivity of N-acetyl-lactosamine synthesis, using the β-galactosidase from Bacillus circulans in hydro-organic media.


    Bridiau, Nicolas; Issaoui, Neyssène; Maugard, Thierry


    The enzymatic synthesis of N-acetyl-lactosamine (LacNAc) by the transgalactosylation of N-acetyl-D-glucosamine (GlcNAc), catalyzed by the β-galactosidase from Bacillus circulans (BcβGal), was studied in hydro-organic media, starting from o-nitrophenyl-β-D-galactopyranoside (oNPG) as a galactosyl donor. Thermal stability and synthesis activity of BcβGal were shown to depend on the organic solvent polarity, characterized by its Log P value. BcβGal was thus most stable in 10% (v/v) t-BuOH, an organic solvent found to have a stabilizing and/or weakly denaturing property, which was confirmed for high t-BuOH concentrations. In the same manner, the optimal synthesis yield increased as the Log P value of the organic solvent increased. The best results were obtained for reactions carried out in 10% (v/v) pyridine or 2-methyl-2-butanol, which gave 47% GlcNAc transgalactosylation yield based on starting oNPG, of which 23% (11 mM; 4.3 g/L) consisted in LacNAc synthesis. Furthermore, it was also established that both the GlcNAc transgalactosylation yield and the enzyme regioselectivity depended on the percentage of organic solvent used, the optimal percentage varying from 10 to 40% (v/v), depending on the solvent. This phenomenon was found to correlate mainly with the thermodynamic activity of water (a(w)) in the aqueous organic solvent mixture, which was found to be optimal when close to 0.96, whatever the organic solvent used. Finally, this study highlighted the fact that the regioselectivity of BcβGal for 1-4 linkage formation could be advantageously managed by controlling the a(w) parameter.

  7. Exogenous surfactant enhances the delivery of recombinant adenoviral vectors to the lung.


    Katkin, J P; Husser, R C; Langston, C; Welty, S E


    Somatic gene therapy for pulmonary diseases must be accomplished in vivo, requiring the spread of a gene transfer vector across a vast expanse of respiratory epithelium. Surfactant, a naturally occurring protein and lipid mixture used to treat the respiratory distress syndrome of prematurity, disperses rapidly and evenly throughout the lung. We employed exogenous bovine surfactant (Survanta beractant) as a carrier vehicle for pulmonary delivery of a recombinant adenovirus expressing beta-galactosidase (beta-Gal). Rats treated with an adenovirus-beractant mixture demonstrated more uniform lobar distribution of transgene expression than rats treated with the same amount of virus in saline. Tissue homogenates were examined for quantitative beta-Gal expression by reaction with o-nitrophenol beta-n-galactopyranoside (ONPG). The degree of beta-Gal activity was affected by both the volume and type of carrier used to deliver the virus. At low volumes (0.5 ml, 1.3 ml/kg), beractant-treated animals demonstrated significantly greater pulmonary beta-Gal activity than saline-treated animals (p < 0.002) and untreated controls. At high volume (1.2 ml, 4 ml/kg), average beta-Gal activity was similar between groups treated with beractant or saline, but was more variable within the saline treated group. Higher volumes of delivery medium were associated with increased levels of beta-Gal expression regardless of the carrier used. Survanta was well tolerated by the animals and did not affect the duration of transgene expression. Exogenous beractant provides a useful medium for delivering recombinant adenoviruses to the lung when diffuse distribution of transgene expression is desired.

  8. Online Monitoring of Enzymatic Reactions Using Time-Resolved Desorption Electrospray Ionization Mass Spectrometry.


    Cheng, Si; Wu, Qiuhua; Xiao, He; Chen, Hao


    Electrospray ionization mass spectrometry (ESI-MS) is powerful for determining enzymatic reaction kinetics because of its soft ionization nature. However, it is limited to use ESI-favored solvents containing volatile buffers (e.g., ammonium acetate). In addition, lack of a quenching step for online ESI-MS reaction monitoring might introduce inaccuracy, due to the possible acceleration of reaction in the sprayed microdroplets. To overcome these issues, this study presents a new approach for online measuring enzymatic reaction kinetics using desorption electrospray ionization mass spectrometry (DESI-MS). By using DESI-MS, enzymatic reaction products in a buffered aqueous media (e.g., a solution containing Tris buffer or high concentration of inorganic salts) could be directly detected. Furthermore, by adjusting the pH and solvent composition of the DESI spray, reaction can be online quenched to avoid the postionization reaction event, leading to fast and accurate measurement of kinetic constants. Reaction time control can be obtained simply by adjusting the injection flow rates of enzyme and substrate solutions. Enzymatic reactions examined in this study include hydrolysis of 2-nitrophenyl-β-D-galactopyranoside by β-galactosidase and hydrolysis of acetylcholine by acetylcholinesterase. Derived Michaelis-Menten constants Km for these two reactions were determined to be 214 μM and 172 μM, respectively, which are in good agreement with the values of 300 μM and 230 μM reported in literature, validating the DESI-MS approach. Furthermore, this time-resolved DESI-MS also allowed us to determine Km and turnover number kcat for trypsin digestion of angiotensin II (Km and kcat are determined to be 6.4 mM and 1.3 s(-1), respectively).

  9. Identification of a dehydrogenase required for lactose metabolism in Caulobacter crescentus.


    Arellano, Benjamin H; Ortiz, Janett D; Manzano, Janet; Chen, Joseph C


    Caulobacter crescentus, which thrives in freshwater environments with low nutrient levels, serves as a model system for studying bacterial cell cycle regulation and organelle development. We examined its ability to utilize lactose (i) to gain insight into the metabolic capacities of oligotrophic bacteria and (ii) to obtain an additional genetic tool for studying this model organism, aiming to eliminate the basal enzymatic activity that hydrolyzes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside (X-gal). Using a previously isolated transposon mutant, we identified a gene, lacA, that is required for growth on lactose as the sole carbon source and for turning colonies blue in the presence of X-gal. LacA, which contains a glucose-methanol-choline (GMC) oxidoreductase domain, has homology to the flavin subunit of Pectobacterium cypripedii's gluconate dehydrogenase. Sequence comparisons indicated that two genes near lacA, lacB and lacC, encode the other subunits of the membrane-bound dehydrogenase. In addition to lactose, all three lac genes are involved in the catabolism of three other beta-galactosides (lactulose, lactitol, and methyl-beta-d-galactoside) and two glucosides (salicin and trehalose). Dehydrogenase assays confirmed that the lac gene products oxidize lactose, salicin, and trehalose. This enzymatic activity is inducible, and increased lac expression in the presence of lactose and salicin likely contributes to the induction. Expression of lacA also depends on the presence of the lac genes, implying that the dehydrogenase participates in induction. The involvement of a dehydrogenase suggests that degradation of lactose and other sugars in C. crescentus may resemble a proposed pathway in Agrobacterium tumefaciens.

  10. Diffusion and reaction in microbead agglomerates.


    Nunes Kirchner, Carolina; Träuble, Markus; Wittstock, Gunther


    Scanning electrochemical microscopy has been used to analyze the flux of p-aminonophenol (PAP) produced by agglomerates of polymeric microbeads modified with galactosidase as a model system for the bead-based heterogeneous immunoassays. With the use of mixtures of enzyme-modified and bare beads in defined ratio, agglomerates with different saturation levels of the enzyme modification were produced. The PAP flux depends on the intrinsic kinetics of the galactosidase, the local availability of the substrate p-aminophenyl-beta-D-galactopyranoside (PAPG), and the external mass transport conditions in the surrounding of the agglomerate and the internal mass transport within the bead agglomerate. The internal mass transport is influenced by the diffusional shielding of the modified beads by unmodified beads. SECM in combination with optical microscopy was used to determine experimentally the external flux. These data are in quantitative agreement with boundary element simulation considering the SECM microelectrode as an interacting probe and treating the Michaelis-Menten kinetics of the enzyme as nonlinear boundary conditions with two independent concentration variables [PAP] and [PAPG]. The PAPG concentration at the surface of the bead agglomerate was taken as a boundary condition for the analysis of the internal mass transport condition as a function of the enzyme saturation in the bead agglomerate. The results of this analysis are represented as PAP flux per contributing modified bead and the flux from freely suspended galactosidase-modified beads. These numbers are compared to the same number from the SECM experiments. It is shown that depending on the enzyme saturation level a different situation can arise where either beads located at the outer surface of the agglomerate dominate the contribution to the measured external flux or where the contribution of buried beads cannot be neglected for explaining the measured external flux.

  11. Flavonoid constituents, cytotoxic and antioxidant activities of Gleditsia triacanthos L. leaves

    PubMed Central

    Mohammed, R.S.; Abou Zeid, A.H.; El Hawary, S.S.; Sleem, A.A.; Ashour, W.E.


    Gleditsia triacanthos L. is a deciduous tree belonging to the family Fabaceae. It possesses important biological activities as anti-mutagenic, anticancer, cytotoxic and treating rheumatoid arthritis. The total ethanol extract (EtOHE) and successive extracts (petroleum ether, chloroform, ethyl acetate, and aqueous ethanol) were prepared from the leaves. Eight flavone glycosides and two flavone aglycones named vicenin-I (1), vitexin (2), isovitexin (3), orientin (4), isoorientin (5), luteolin-7-O-ß-glucopyranoside (6), luteolin-7-O-ß-galactopyranoside (7), apigenin-7-O-ß-glucopyranoside (8), luteolin (9) and apigenin (10) were isolated from the aqueous ethanol extract of G. triacanthos L. leaves. Potent cytotoxic activity of the EtOHE extract was observed against the liver (IC50 = 1.68 μg), breast (IC50 = 0.74 μg), cervix (IC50 = 1.28 μg), larynx (IC50 = 0.67 μg) and colon (IC50 = 2.50 μg) cancer cell lines. Cytotoxic activity of compounds 2, 4, 6 and 8 against, the liver, breast and colon cancer cell lines was also proved. Evaluation of the in-vivo antioxidant activity of the EtOHE and successive extracts revealed that the highest activity was exhibited by 100 mg of EtOHE (97.89% potency) as compared with vitamin E (100% potency). Compound 6 showed 91.8% free radical scavenging activity. PMID:25473363

  12. Relationship between the composition of flavonoids and flower colors variation in tropical water lily (Nymphaea) cultivars.


    Zhu, Manlan; Zheng, Xuchen; Shu, Qingyan; Li, Hui; Zhong, Peixing; Zhang, Huijin; Xu, Yanjun; Wang, Lijin; Wang, Liangsheng


    Water lily, the member of the Nymphaeaceae family, is the symbol of Buddhism and Brahmanism in India. Despite its limited researches on flower color variations and formation mechanism, water lily has background of blue flowers and displays an exceptionally wide diversity of flower colors from purple, red, blue to yellow, in nature. In this study, 34 flavonoids were identified among 35 tropical cultivars by high-performance liquid chromatography (HPLC) with photodiode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS). Among them, four anthocyanins: delphinidin 3-O-rhamnosyl-5-O-galactoside (Dp3Rh5Ga), delphinidin 3-O-(2"-O-galloyl-6"-O-oxalyl-rhamnoside) (Dp3galloyl-oxalylRh), delphinidin 3-O-(6"-O-acetyl-β-glucopyranoside) (Dp3acetylG) and cyanidin 3- O-(2"-O-galloyl-galactopyranoside)-5-O-rhamnoside (Cy3galloylGa5Rh), one chalcone: chalcononaringenin 2'-O-galactoside (Chal2'Ga) and twelve flavonols: myricetin 7-O-rhamnosyl-(1 → 2)-rhamnoside (My7RhRh), quercetin 7-O-galactosyl-(1 → 2)-rhamnoside (Qu7GaRh), quercetin 7-O-galactoside (Qu7Ga), kaempferol 7-O-galactosyl-(1 → 2)-rhamnoside (Km7GaRh), myricetin 3-O-galactoside (My3Ga), kaempferol 7-O-galloylgalactosyl-(1 → 2)-rhamnoside (Km7galloylGaRh), myricetin 3-O-galloylrhamnoside (My3galloylRh), kaempferol 3-O-galactoside (Km3Ga), isorhamnetin 7-O-galactoside (Is7Ga), isorhamnetin 7-O-xyloside (Is7Xy), kaempferol 3-O-(3"-acetylrhamnoside) (Km3-3"acetylRh) and quercetin 3-O-acetylgalactoside (Qu3acetylGa) were identified in the petals of tropic water lily for the first time. Meanwhile a multivariate analysis was used to explore the relationship between pigments and flower color. By comparing, the cultivars which were detected delphinidin 3-galactoside (Dp3Ga) presented amaranth, and detected delphinidin 3'-galactoside (Dp3'Ga) presented blue. However, the derivatives of delphinidin and cyanidin were more complicated in red group. No anthocyanins were detected within white and

  13. Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.


    Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef


    Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is

  14. High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.


    Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun


    Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

  15. Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species.

    PubMed Central

    Merlino, J; Siarakas, S; Robertson, G J; Funnell, G R; Gottlieb, T; Bradbury, R


    A new chromogenic plate medium, CHROMagar Orientation, was evaluated for use in the differentiation and presumptive identification of gram-negative bacilli and Enterococcus species by a multipoint inoculation (replicator) technique. In this study, 1,404 gram-negative bacilli and 74 enterococcal isolates were tested on CHROMagar Orientation. Six control American Type Culture Collection strains were also included with the testing to ensure quality control of the media. Of the Escherichia coli isolates (n = 588) tested, 99.3% produced a pink-to-red color. Only in four isolates that were O-nitrophenyl-beta-D-galactopyranoside (ONPG) negative did this result differ. Proteus mirabilis and P. vulgaris were well differentiated on this medium. P. mirabilis (n = 184) produced a clear colony with diffusible brown pigment around the periphery. By contrast, 15 of 16 P. vulgaris isolates produced bluish-green colonies with a slight brown background. All Aeromonas hydrophila isolates (n = 26) tested produced clear to pink colonies at 35 to 37 degrees C. This colony color changed to blue after 2 to 3 h of incubation at room temperature. A. hydrophila exhibited stronger color and better growth at 30 degrees C. Serratia marcescens (n = 29) demonstrated an aqua blue color that deepened to a darker blue when exposed to room temperature. All enterococcal isolates (n = 74) resulted in a blue color and gave pinpoint colonies on purity subcultures at 35 to 37 degrees C after 18 h of incubation. Similarity in color resulted in failure to discriminate accurately between Klebsiella, Enterobacter, and Citrobacter species. However, these species could be readily differentiated from other members of the family Enterobacteriaceae. Pseudomonas aeruginosa (n = 151) was easily differentiated from members of the Enterobacteriaceae but was less easily distinguishable from other gram-negative nonmembers of the Enterobacteriaceae. The medium was found to facilitate easy visual detection of mixed

  16. The Gonococcal NlpD Protein Facilitates Cell Separation by Activating Peptidoglycan Cleavage by AmiC

    PubMed Central

    Stohl, Elizabeth A.; Lenz, Jonathan D.; Dillard, Joseph P.


    ABSTRACT Key steps in bacterial cell division are the synthesis and subsequent hydrolysis of septal peptidoglycan (PG), which allow efficient separation of daughter cells. Extensive studies in the Gram-negative, rod-shaped bacterium Escherichia coli have revealed that this hydrolysis is highly regulated spatially and temporally. Neisseria gonorrhoeae is an obligate Gram-negative, diplococcal pathogen and is the only causative agent of the sexually transmitted infection gonorrhea. We investigated how cell separation proceeds in this diplococcal organism. We demonstrated that deletion of the nlpD gene in strain FA1090 leads to poor growth and to an altered colony and cell morphology. An isopropyl-beta-d-galactopyranoside (IPTG)-regulated nlpD complemented construct can restore these defects only when IPTG is supplied in the growth medium. Thin-section transmission electron microscopy (TEM) revealed that the nlpD mutant strain grew in large clumps containing live and dead bacteria, which was consistent with deficient cell separation. Biochemical analyses of purified NlpD protein showed that it was able to bind purified PG. Finally, we showed that, although NlpD has no hydrolase activity itself, NlpD potentiates the hydrolytic activity of AmiC. These results indicate that N. gonorrhoeae NlpD is required for proper cell growth and division through its interactions with the amidase AmiC. IMPORTANCE N. gonorrhoeae is the sole causative agent of the sexually transmitted infection gonorrhea. The incidence of antibiotic-resistant gonococcal infections has risen sharply in recent years, and N. gonorrhoeae has been classified as a “superbug” by the CDC. Since there is a dearth of new antibiotics to combat gonococcal infections, elucidating the essential cellular process of N. gonorrhoeae may point to new targets for antimicrobial therapies. Cell division and separation is one such essential process. We identified and characterized the gonococcal nlpD gene and showed that

  17. Synthesis of branched dithiotrisaccharides via ring-opening reaction of sugar thiiranes.


    Repetto, Evangelina; Manzano, Verónica E; Uhrig, María Laura; Varela, Oscar


    Satisfactory procedures are described for the synthesis of 5,6- and 3,4-thiirane derivatives from the respective hexofuranose or hexopyranose epoxide precursors. The controlled ring-opening reaction of thiiranes by 1-thioaldoses was successfully accomplished to afford, regio- and stereoselectively, β-S-(1→4)-3,4-dithiodisaccharides. For instance, the regioselective attack of per-O-acetyl-1-thioglucose (16) to C-4 of 2-propyl 2,6-di-O-acetyl-3,4-epithio-α-D-galactopyranoside (14) gave the derivative of Glcp-β-S-(1→4)-3,4-dithioGlcp-O-iPr (17). This thiodisaccharide was accompanied by the (1→3)-disulfide 18, formed between 16 and 17, and the symmetric (3→3)-disulfide 19, which resulted from the oxidative dimerization of 17. However, the S-acetyl derivative of 17 could be obtained in good yield (62%) by LiAlH(4) reduction of the crude mixture 17-19, followed by acetylation. The same sequence of reactions starting from 14 and the 1-thiolate of Galp afforded the per-O,S-acetyl derivative of Galp-β-S-(1→4)-3,4-dithio-α-D-Glcp-O-iPr (23), which was selectively S-deacetylated to give 25. The dithiosaccharides 17 and 25 are 3,4-di-S-analogues of derivatives of the natural disaccharides cellobiose and lactose, respectively. The ring-opening reaction of 5,6-epithiohexofuranoses of D-galacto (8) or L-altro (11) configuration with 1-thioaldoses was also regio- and stereoselective to give the respective β-S-(1→6)-linked 5,6-dithiodisaccharides 26 or 29 in excellent yields. Glycosylation of the free thiol group of 17, 25, or 26, using trichloroacetimidates as glycosyl donors, led to the corresponding branched dithiotrisaccharides. Some of them are sulfur analogues of derivatives of branched trisaccharides found in natural polysaccharides.

  18. Bioconcentration of the intense sweetener sucralose in a multitrophic battery of aquatic organisms.


    Lillicrap, Adam; Langford, Katherine; Tollefsen, Knut Erik


    Reports of the intense (artificial) sweetener sucralose (1,6-dichloro-1,6-dideoxy-β-D-fructo-furanosyl 4-chloro-4-deoxy-α-D-galactopyranoside) in various environmental compartments have led to speculations about biological effects in nontarget species living in areas receiving discharges from anthropogenic activities. We have, as the first step in the risk assessment of this compound, conducted bioaccumulation studies in the freshwater alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna, and zebrafish (Danio rerio). The freshwater algae and the daphnid tests were performed using a 48-h static exposure system, whereas the zebrafish test was performed using a 48-h semi static exposure system followed by 48 h flow-through of clean water for the depuration phase. All three studies were conducted with two exposure concentrations (10 and 100 mg/L), and the concentrations of sucralose in water and biota were verified by liquid chromatography/mass spectrometry. The studies showed that uptake of sucralose was assumed to achieve a steady state within the first 48 h, and the bioconcentration factor at the assumed steady state (BCF(SS) ) was calculated to be less than 1 for algae and between 1.6 to 2.2 for the daphnids. The fish BCF(SS), assumed to occur between 24 to 48 hours, were calculated to be less than 1 for both concentrations tested. A first-order one-compartment (uptake phase) and a first-order two-compartment (elimination phase) model characterized the uptake and depuration kinetics in zebrafish (k(1)=0.027-0.038/h and k(2)=0.206-0.222/h, t(95)=13.5 to 14.6 h, t(50)=3.1 to 3.3 h, and BCF(kinetic)=0.4 to 0.9). The current study shows that sucralose does not bioaccumulate in aquatic organisms from different tiers of the food web, and that the BCF's obtained were considerably lower than the criteria set to identify persistent, bioaccumulative, and toxic substances (i.e., BCF ≥ 2,000).

  19. New Isorhamnetin Derivatives from Salsola imbricata Forssk. Leaves with Distinct Anti-inflammatory Activity

    PubMed Central

    Osman, Samir M.; El Kashak, Walaa A.; Wink, Michael; El Raey, Mohamed A.


    Background: Salsola imbricata Forssk. is a shrub widely growing in Egypt, used as a camel food, traditionally, used as anti-inflammatory agent. Literature survey showed no report about the anti-inflammatory activity of S. imbricata. Aim of the Study: This work was designed to study the phenolic constituents and to provide evidence for the traditional use of S. imbricata as an anti-inflammatory agent. Materials and Methods: The in vitro anti-inflammatory activity of the total aqueous methanol extract and some isolated compounds were investigated in RAW 264.7 macrophage cells using nitric oxide assay. All chemical structures were identified on the basis of electrospray ionization-mass spectrometry, one- and two-dimension nuclear magnetic resonance. Results: Nine phenolic compounds, among them two new natural products; isorhamnetin-3-O-β-D-glucuronyl (1’’’→4’’) glucuronide (1) and its dimethyl ester; isorhamnetin-3-O-β-D-di glucuronate dimethyl ester (2), two isorhamnetin glycosides: Isorhamnetin-3-O-β-D-galactopyranoside (3), isorhamnetin-3-O-β-D-glucopyranoside (4), and isorhamnetin (5). In addition, an alkaloidal phenolic; trans N-feruloyl tyramine (6), three phenolic acids: Isovanillic acid (7), ferulic acid (8), and p-hydroxy benzoic acid (9) were isolated from salsola imbricata leaves. All compounds were isolated and identified for the first time from this plant except compound (6). The extract and the tested compounds showed distintict anti-inflammatory activities with no toxicity on RAW 264.7 macrophage cells. Conclusion: The extract and the tested compounds showed distintict anti-inflammatory activities with no toxicity on RAW 264.7 macrophage cells. SUMMARY Investigation of the chemical constituents of the leaves of Salsola imbricata led to isolation of two new isorhamnetin derivatives: isorhamnetin.3-O-β-D.glucuronyl (1’“→”) glucuronide (1) and its dimethyl ester (2), together with seven known phenolic compounds. The extract and the

  20. Identification and characterization of a novel secreted glycosidase with multiple glycosidase activities in Streptococcus intermedius.


    Imaki, Hidenori; Tomoyasu, Toshifumi; Yamamoto, Naoki; Taue, Chiharu; Masuda, Sachiko; Takao, Ayuko; Maeda, Nobuko; Tabata, Atsushi; Whiley, Robert A; Nagamune, Hideaki


    Streptococcus intermedius is a known human pathogen and belongs to the anginosus group (S. anginosus, S. intermedius, and S. constellatus) of streptococci (AGS). We found a large open reading frame (6,708 bp) in the lac operon, and bioinformatic analysis suggested that this gene encodes a novel glycosidase that can exhibit β-d-galactosidase and N-acetyl-β-d-hexosaminidase activities. We, therefore, named this protein "multisubstrate glycosidase A" (MsgA). To test whether MsgA has these glycosidase activities, the msgA gene was disrupted in S. intermedius. The msgA-deficient mutant no longer showed cell- and supernatant-associated β-d-galactosidase, β-d-fucosidase, N-acetyl-β-d-glucosaminidase, and N-acetyl-β-d-galactosaminidase activities, and all phenotypes were complemented in trans with a recombinant plasmid carrying msgA. Purified MsgA had all four of these glycosidase activities and exhibited the lowest Km with 4-methylumbelliferyl-linked N-acetyl-β-d-glucosaminide and the highest kcat with 4-methylumbelliferyl-linked β-d-galactopyranoside. In addition, the purified LacZ domain of MsgA had β-d-galactosidase and β-d-fucosidase activities, and the GH20 domain exhibited both N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities. The β-d-galactosidase and β-d-fucosidase activities of MsgA are thermolabile, and the optimal temperature of the reaction was 40°C, whereas almost all enzymatic activities disappeared at 49°C. The optimal temperatures for the N-acetyl-β-d-glucosaminidase and N-acetyl-β-d-galactosaminidase activities were 58 and 55°C, respectively. The requirement of sialidase treatment to remove sialic acid residues of the glycan branch end for glycan degradation by MsgA on human α1-antitrypsin indicates that MsgA has exoglycosidase activities. MsgA and sialidase might have an important function in the production and utilization of monosaccharides from oligosaccharides, such as glycans for survival in a normal

  1. Design of a coil satellite centrifuge and its performance on counter-current chromatographic separation of 4-methylumbelliferyl sugar derivatives with polar organic-aqueous two-phase solvent systems.


    Shinomiya, Kazufusa; Tokura, Koji; Kimura, Emiru; Takai, Midori; Harikai, Naoki; Yoshida, Kazunori; Yanagidaira, Kazuhiro; Ito, Yoichiro


    A new high-speed counter-current chromatograph, named coil satellite centrifuge (CSC), was designed and fabricated in our laboratory. The CSC apparatus produces the satellite motion such that the coiled column simultaneously rotates around the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (the central axis of the column) (ω3). In order to achieve this triplicate rotary motion without twisting of the flow tube, the rotation of each axis was determined by the following formula: ω1=ω2+ω3. This relation enabled to lay out the flow tube without twisting by the simultaneous rotation of three axes. The flow tube was introduced from the bottom side of the apparatus into the sun axis of the first rotary frame reaching the upper side of the planet axis and connected to the column in the satellite axis. The performance of the apparatus was examined on separation of 4-methylumbelliferyl (MU) sugar derivatives as test samples with organic-aqueous two-phase solvent systems composed of ethyl acetate/1-butanol/water (3:2:5, v/v) for lower phase mobile and (1:4:5, v/v) for upper phase mobile. With lower phase mobile, five 4-MU sugar derivatives including β-D-cellobioside (Cel), β-D-glucopyranoside, α-D-mannopyranoside, β-D-fucopyranoside and α-L-fucopyranoside (α-L-Fuc) were separated with the combined rotation around each axis at counterclockwise (CCW) (ω1) - CCW (ω2) - CCW (ω3) by the flow tube distribution. With upper phase mobile, three 4-MU sugar derivatives including α-L-Fuc, β-D-galactopyranoside and Cel were separated with the combined rotation around each axis at clockwise (CW) (ω1) - CW (ω2) - CW (ω3) by the flow tube distribution. A series of experiments on peak resolution and stationary phase retention revealed that better partition efficiencies were obtained at the flow rate of 0.5 mL/min (column 1) and 0.8 mL/min (column 2) for lower phase mobile and 0.2 mL/min (column 1) and 0.4 mL/min (column 2) for upper phase

  2. Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

    PubMed Central

    Sahin-Tóth, M.; Kaback, H. R.


    Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+. PMID

  3. A biosensor platform for rapid detection of E. coli in drinking water.


    Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza


    There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or

  4. Development of selective and differential medium for Shigella sonnei using three carbohydrates (lactose, sorbitol, and xylose) and X-Gal.


    Na, G N; Kim, S A; Kwon, O C; Rhee, M S


    The aim of this study was to develop a new selective and differential medium for isolating Shigella sonnei (designated 3SD medium). The new medium was based on three carbohydrates (lactose, sorbitol, and xylose) and a chromogenic substrate (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, X-Gal). S. sonnei cannot ferment lactose, sorbitol, or xylose, but can ferment X-Gal, which generates turquoise-blue colonies with rough edges. Other bacteria (54 strains of foodborne pathogens and spoilage bacteria) produced visually distinct colonies on 3SD medium (colorless or pink-violet colonies), or their growth was inhibited on 3SD medium. The optimum concentration of 50 mg/L X-Gal was selected because it yielded the highest level of morphological discrimination between S. sonnei and other bacteria, and this concentration was cost-effective. Bile salt concentration optimization was performed using healthy, heat-injured, and acid-injured S. sonnei. The recovery rate differed significantly depending on the bile salt concentration; media containing >1.0 g/L bile salt showed significantly lower recovery of stress-injured cells than medium containing 0.5 g/L bile salt (P<0.05). Growth of all Gram-positive bacteria was inhibited on medium containing 0.5 g/L bile salt; therefore, this concentration was used as the optimal concentration. Previous media used to isolate Shigella spp. (MacConkey, xylose lysine desoxycholate, and Salmonella-Shigella agar) showed poor performance when used to support the growth of injured S. sonnei cells, whereas 3SD medium supported a high growth rate of injured and healthy cells (equivalent to that obtained with nutrient-rich tryptic soy agar). To validate the performance of 3SD medium with real specimens, S. sonnei and other bacteria were spiked into samples such as untreated water, carrot, salad, and oyster. 3SD medium showed superior specificity (100%) and sensitivity (100%) for S. sonnei, and yielded no false-positive or false-negative results

  5. Aeromonas jandaei (formerly genospecies DNA group 9 A. sobria), a new sucrose-negative species isolated from clinical specimens.

    PubMed Central

    Carnahan, A; Fanning, G R; Joseph, S W


    A large numerical taxonomy study conducted in 1988 of 165 mostly clinical Aeromonas strains from diverse geographic sources produced a cluster (S = 84%, SSM) of four sucrose-negative strains that included the DNA definition strain for DNA group 9 A. sobria (CDC 0787-80). These four strains, together with five additional strains received in 1989, were subjected to DNA-DNA hybridization (hydroxyapatite, 32P, 60 and 75 degrees C), and all eight strains were closely related to the ninth labeled DNA group 9 definition strain CDC 0787-80 (73 to 86% relatedness at 60 degrees C and 68 to 80% relatedness at 75 degrees C; percent divergence, 2.0 to 3.5). Type strains and DNA definition strains for all other established Aeromonas species were only 35 to 72% related (60 degrees C) to CDC 0787-80. We propose the name Aeromonas jandaei for this highly related group of nine strains, formerly known as DNA group 9 A. sobria. The type strain was designated ATCC 49568 (CDC 0787-80). The nine strains were examined at 36 degrees C and were found to be resistant to 0/129 (vibriostatic agent) and uniformly positive for oxidase, gas production from glucose, indole, lysine decarboxylase, arginine dihydrolase, o-nitrophenyl-beta-D-galactopyranoside, motility (25 degrees C), nitrate reduction, citrate utilization, hemolysis on sheep blood agar, and growth in Trypticase soy broth with no added NaCl. They all fermented D-glucose, D-mannitol, and mannose but did not ferment sucrose, cellobiose, L-arabinose, inositol, salicin, or D-sorbitol. They were uniformly negative for esculin and urea hydrolysis, elastase production, ornithine decarboxylation, and the string test. The antibiogram of A. jandaei resembled that of other aeromonads (resistance to ampicillin and cephalothin), but it differed from most other aeromonads because of resistance to single dilution of colistin and differed from clinical A. veronii biogroup sorbria (formerly A. sobria) by its nearly uniform resistance to cephalothin

  6. Design of a novel coil satellite centrifuge and its performance on counter-current chromatographic separation of 4-methylumbelliferyl sugar derivatives with organic-aqueous two-phase solvent systems

    PubMed Central

    Shinomiya, Kazufusa; Tokura, Koji; Kimura, Emiru; Takai, Midori; Harikai, Naoki; Yoshida, Kazunori; Yanagidaira, Kazuhiro; Ito, Yoichiro


    A new high-speed counter-current chromatograph, named coil satellite centrifuge (CSC), was designed and fabricated in our laboratory. The CSC apparatus produces the satellite motion such that the coiled column simultaneously rotates around the sun axis (the angular velocity, ω1), the planet axis (ω2) and the satellite axis (the central axis of the column) (ω3). In order to achieve this triplicate rotary motion without twisting of the flow tube, the rotation of each axis was determined by the following formula: ω1 = ω2 + ω3. This relation enabled to lay out the flow tube by two different ways, the SS type and the JS type. In the SS type, the flow tube was introduced from the upper side of the apparatus into the sun axis of the first rotary frame and connected to the planet axis of the second rotary frame like a double letter SS. In the JS type, the flow tube was introduced from the bottom of the apparatus into the sun axis reaching the upper side of the planet axis an inversed letter J, followed by distribution as in the SS type. The performance of the apparatus was examined on separation of 4-methylumbelliferyl (MU) sugar derivatives as test samples with organic-aqueous two-phase solvent systems composed of ethyl acetate/1-butanol/water (3 : 2 : 5, v/v) for lower phase mobile and (1 : 4 : 5, v/v) for upper phase mobile. With lower phase mobile, five 4-MU sugar derivatives including β-D-cellobioside (Cel), β-D-glucopyranoside, α-D-mannopyranoside, β-D-fucopyranoside and α-L-fucopyranoside (α-L-Fuc) were separated with the combined rotation around each axis at counterclockwise (CCW) (ω1) – CCW (ω2) – CCW (ω3) by the JS type flow tube distribution. With upper phase mobile, three 4-MU sugar derivatives including α-L-Fuc, β-D-galactopyranoside and Cel were separated with the combined rotation around each axis at clockwise (CW) (ω1) – CW (ω2) – CW (ω3) by the JS type flow tube distribution. A series of experiments on peak resolution and

  7. Trabulsiella guamensis, a new genus and species of the family Enterobacteriaceae that resembles Salmonella subgroups 4 and 5.


    McWhorter, A C; Haddock, R L; Nocon, F A; Steigerwalt, A G; Brenner, D J; Aleksić, S; Bockemühl, J; Farmer, J J


    In 1985 the vernacular name Enteric Group 90 was coined for a small group of strains that had been referred to our laboratory as probable strains of Salmonella but did not agglutinate in Salmonella typing antisera. By DNA-DNA hybridization (hydroxyapatite method, 32P), seven strains of Enteric Group 90 were found to be closely related (98 to 100% at 60 degrees C and 94 to 100% at 75 degrees C) to the first strain received (0370-85). The relatedness of Enteric Group 90 to 62 strains of other species of the family Enterobacteriaceae was only 6 to 41%, with the highest values obtained with strains of Salmonella, Kluyvera, Shigella, Klebsiella, Enterobacter, and Citrobacter. We propose a new genus, Trabulsiella, with a single new species, Trabulsiella guamensis, for the highly related group of eight strains formerly known as Enteric Group 90. The type strain is designated ATCC 49490 (CDC 0370-85). T. guamensis strains grew well at 36 degrees C and had positive reactions in the following tests: methyl red, citrate utilization (Simmons) (38% positive at day 1, 88% positive at 2 days), H2S production, lysine decarboxylase, arginine dihydrolase (50% positive at 2 days, 100% positive at 7 days), ornithine decarboxylase, motility, growth in KCN medium, mucate fermentation, acetate utilization, nitrate reduction to nitrite, weak tyrosine hydrolysis (88% positive at 2 days, 100% positive at 7 days), and ONPG (o-nitrophenyl-beta-D-galactopyranoside) test. The strains fermented D-glucose with gas production and fermented L-arabinose, cellobiose, D-galactose, D-galacturonate, maltose, D-mannitol, D-mannose, L-rhamnose, D-sorbitol, trehalose, and D-xylose. T. guamensis strains had negative reactions in the following tests: indole production (13% positive), Voges-Proskauer, urea hydrolysis, phenylalanine deaminase, malonate utilization, lipase (corn oil), DNase, oxidase, pigment production, and acid production from adonitol, D-arabitol, dulcitol, erythritol, myo-inositol, melibiose

  8. Screening Mixtures of Small Molecules for Binding to Multiple Sites on the Surface Tetanus Toxin C Fragment by Bioaffinity NMR

    SciTech Connect

    Cosman, M; Zeller, L; Lightstone, F C; Krishnan, V V; Balhorn, R


    also contains 3-sialyllactose (another predicted site 1 binder) and bisbenzimide 33342 (non-binder). A series of five predicted Site-2 binders were then screened sequentially in the presence of the Site-1 binder doxorubicin. These experiments showed that the compounds lavendustin A and naphthofluorescein-di-({beta}-D-galactopyranoside) binds along with doxorubicin to TetC. Further experiments indicate that doxorubicin and lavendustin are potential candidates to use in preparing a bidendate inhibitor specific for TetC. The simultaneous binding of two different predicted Site-2 ligands to TetC suggests that they may bind multiple sites. Another possibility is that the conformations of the binding sites are dynamic and can bind multiple diverse ligands at a single site depending on the pre-existing conformation of the protein, especially when doxorubicin is already bound.

  9. Structure-specificity relationships in Abp, a GH27 β-L-arabinopyranosidase from Geobacillus stearothermophilus T6.


    Lansky, Shifra; Salama, Rachel; Solomon, Hodaya V; Feinberg, Hadar; Belrhali, Hassan; Shoham, Yuval; Shoham, Gil


    L-Arabinose sugar residues are relatively abundant in plants and are found mainly in arabinan polysaccharides and in other arabinose-containing polysaccharides such as arabinoxylans and pectic arabinogalactans. The majority of the arabinose units in plants are present in the furanose form and only a small fraction of them are present in the pyranose form. The L-arabinan-utilization system in Geobacillus stearothermophilus T6, a Gram-positive thermophilic soil bacterium, has recently been characterized, and one of the key enzymes was found to be an intracellular β-L-arabinopyranosidase (Abp). Abp, a GH27 enzyme, was shown to remove β-L-arabinopyranose residues from synthetic substrates and from the native substrates sugar beet arabinan and larch arabinogalactan. The Abp monomer is made up of 448 amino acids, and based on sequence homology it was suggested that Asp197 is the catalytic nucleophile and Asp255 is the catalytic acid/base. In the current study, the detailed three-dimensional structure of wild-type Abp (at 2.28 Å resolution) and its catalytic mutant Abp-D197A with (at 2.20 Å resolution) and without (at 2.30 Å resolution) a bound L-arabinose product are reported as determined by X-ray crystallography. These structures demonstrate that the three-dimensional structure of the Abp monomer correlates with the general fold observed for GH27 proteins, consisting of two main domains: an N-terminal TIM-barrel domain and a C-terminal all-β domain. The two catalytic residues are located in the TIM-barrel domain, such that their carboxylic functional groups are about 5.9 Å from each other, consistent with a retaining mechanism. An isoleucine residue (Ile67) located at a key position in the active site is shown to play a critical role in the substrate specificity of Abp, providing a structural basis for the high preference of the enzyme towards arabinopyranoside over galactopyranoside substrates. The crystal structure demonstrates that Abp is a tetramer

  10. From Vibrational Spectroscopy to Force Fields and Structures of Saccharides: New Computational Algorithms and Applications

    SciTech Connect

    Pincu, Madeleine; Gerber, Robert Benny


    This work was undertaken with the main objective to investigate basic reactions that take place in relatively simple saccharides (mono-saccharides and cellobiose - the building block of cellulose) , in isolation and in cluster with few water molecules or with (gas-phase) clusters of few waters and ionic compounds (salt, isolated ions like H{sup +} or OH{sup -}). Within the context of this work, different potentials were investigated; among them, were the PM3 semi empirical potential, DFT/BLYP and a new hybrid potential constructed from MP2 for the harmonic part and from adjusted Hartree-Fock anharmonic interactions (VSCF-PT2). These potentials were evaluated by comparison with experimental data from published sources and from several collaborating groups. The findings show excellent agreement between experiments and predictions with the hybrid VSCF-PT2 potential and very good agreement with predictions obtained from dynamics with dispersion corrected DFT/BLYP potential. Investigation of hydration of cellobiose, was another topic of interest. Guided by a hydration motif demonstrated by our experimental collaborators (team of Prof J.P. Simons), we demonstrated large energetic and structural differences between the two species of cellobiose: cis and trans. The later, which is dominant in solid and liquid phases, is higher in energy in the gas-phase and compared to pure water, it does not disturb as much the network of H bonds. In contrast, the cis species exhibits asymmetric hydration in cluster with up to 25 waters, indicating that it has surfactant properties. Another highlight of this research effort was the successful first time spectrometric and spectroscopic study of a gas-phase protonated sugar derivative (alpha-D-Galactopyranoside) and its interpretation by Ab Initio molecular dynamics (AIMD) simulations. The findings demonstrate the formation of a motif in which a proton bridges between two Oxygen atoms (belonging to OH groups) at the sugar; The vibrational

  11. [Fermentation and purification of recombinant alpha-galactosidase from Pichia pastoris].


    Gao, Xin; Yang, Jun; Li, Su-Bo; Liu, Ze-Peng; Zhang, Yang-Pei


    In order to obtain an adequate supply of alpha-galactosidase for research and practical use, the fermentation, purification and identification of the recombinant coffee bean a-galactosidase were carried out. Baffled flasks containing 100mL BMGY were inoculated with the pPIC9K-Gal/GS115 strain and allowed to grow at 30 degrees C, 250- 300r/min until a maximum optical density at 600nm (OD600) between 2.0 to 6.0 was attained. Entire 400 mL seed culture was transferred aseptically to the 5-liter fermenter, which contained 4 liter sterilized basal salts medium and 4% glycerol. The batch culture grew at 30 degrees C, pH 5.0 until the glycerol was completely consumed, and a glycerol feed was initiated to increase the cell biomass prior to induction with methanol. The culture was centrifuged at 8000 x g and the supernatant was collected. Following ultrafiltration, the retentate was balanced in 20 mmol/L sodium formicate buffer, pH 3.8 and loaded onto a cation-exchange column, HiTrap SP. The column was washed with the same buffer and bound proteins were eluted with 1 mol/L NaCl. The fractions containing recombinant a-galactosidase were pooled and concentrated with PEG20 000. Subsequently, the biochemical properties of the enzyme were determined with typical methods. At last, the fresh human blood A and B erythrocytes were incubated with the purified alpha-galactosidase at 26 degrees C for 2 4 hours. Hemagglutinins were assayed by the standard method. After an elapsed fermentation times (EFT) of 18h, the fed-batch phase was initiated to increase the cell biomass. A cellular yield of nearly 200 g/liter wet cells was achieved when induction was initiated. 72h later, the alpha-galactosidase activity against artificial substrate PNPG (PNP-alpha-galactopyranoside) achieved 36 000u per liter culture. The crude fementation supernatant contained few impurities as detected by SDS-PAGE. The supernatant was purified by cation-exchange chromatography, the target alpha-galactosidase was