Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32–44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated β-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.     

A novel xyloglucan-specific endo-β-1,4-glucanase: biochemical properties and inhibition studies

A novel xyloglucan-specific endo-β-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of ~90 amino acid residues with unknown function. The catalytic domain assumed an (α/β)₈-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis–Menten kinetics, yielding K _m and V _max of 3.61 ± 0.23 mg/ml and 0.30 ± 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K _i of 1.46 ± 0.13 mM. The enzyme was devoid of transglycosylase activities.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Localization of ion-regulatory epithelia in embryos and hatchlings of two cephalopods

The tissue distribution and ontogeny of Na⁺/K⁺-ATPase has been examined as an indicator for ion-regulatory epithelia in whole animal sections of embryos and hatchlings of two cephalopod species: the squid Loligo vulgaris and the cuttlefish Sepia officinalis. This is the first report of the immunohistochemical localization of cephalopod Na⁺/K⁺-ATPase with the polyclonal antibody α (H-300) raised against the human α1-subunit of Na⁺/K⁺-ATPase. Na⁺/K⁺-ATPase immunoreactivity was observed in several tissues (gills, pancreatic appendages, nerves), exclusively located in baso-lateral membranes lining blood sinuses. Furthermore, large single cells in the gill of adult L. vulgaris specimens closely resembled Na⁺/K⁺-ATPase-rich cells described in fish. Immunohistochemical observations indicated that the amount and distribution of Na⁺/K⁺-ATPase in late cuttlefish embryos was similar to that found in juvenile and adult stages. The ion-regulatory epithelia (e.g., gills, excretory organs) of the squid embryos and paralarvae exhibited less differentiation than adults. Na⁺/K⁺-ATPase activities for whole animals were higher in hatchlings of S. officinalis (157.0 ± 32.4 μmol g_FM⁻¹ h⁻¹) than in those of L. vulgaris (31.8 ± 3.3 μmol g_FM⁻¹ h⁻¹). S. officinalis gills and pancreatic appendages achieved activities of 94.8 ± 18.5 and 421.8 ± 102.3 μmol_ATP g_FM⁻¹ h⁻¹, respectively. High concentrations of Na⁺/K⁺-ATPase in late cephalopod embryos might be important in coping with the challenging abiotic conditions (low pH, high pCO₂) that these organisms encounter inside their eggs. Our results also suggest a higher sensitivity of squid vs. cuttlefish embryos to environmental acid-base disturbances.     

Inhibition of CYP3A4 and CYP2C9 by podophyllotoxin: Implication for clinical drug–drug interactions

Podophyllotoxin (PPT) and its derivatives exert significant anti-cancer activities, and one derivative etoposide is often utilized to treat various cancers in the clinic. The aim of the present study is to investigate the inhibitory effects of PPT on major cytochrome P450 (CYP) isoforms in human livers. Inhibition of CYP3A4, CYP2C9, CYP2C8, CYP2D6, CYP2E1 and CYP2A6 by PPT was investigated in the human liver microsomal system. Time-dependent inhibition of CYP3A4 by PPT was also evaluated. The results showed that PPT strongly exhibited inhibitory effects on CYP3A4 and CYP2C9 in a concentration-dependent manner. Half inhibition concentration (IC₅₀) was 1.1 ± 0.3 and 4.6 ± 0.3 μM for CYP3A4 and CYP2C9, respectively. Inhibition kinetic analysis showed that PPT exhibited competitive inhibition towards CYP3A4 and CYP2C9 with K_i of 1.6 and 2.0 μM, respectively. Additionally, PPT exerted time-dependent inhibition towards CYP3A4 and the kinetic parameters were 4.4 ± 2.1 μM and 0.06 ± 0.01 min^–1 for K_I and k_inact, respectively. Our experimental data indicate that potential drug–drug interaction (DDI) might exist when PPT is co-administered with the substrates which mainly undergo CYP3A4- or CYP2C9-mediated metabolism.     

Kinetics and characteristics of 70 °C, VFA-grown, UASB granular sludge

We studied in batch reactors the kinetics and characterization of 70 °C, volatile fatty acids (VFAs)-grown, upflow anaerobic sludge blanket granular sludge with 55 and 35 °C sludge as reference. The half-saturation constant (K _s), the inhibition constant (K _i), the maximum specific methane production rate (μ_CH4max), and the inhibition response coefficient (n) of the 70 °C sludge were 6.15 mM, 48.2 mM, 0.132 h⁻¹, and 2.48, respectively, while no inhibition occurred at 55 and 35 °C, where the K _s was 3.67 and 3.82 mM, respectively. At 70 °C, the highest initial specific methanogenic activity (ISMA, 0.311 gCH₄-COD per gram volatile solids per day) on VFAs was about 12–15% lower than that on acetate and three to four times less than the ISMA for the 55 and 35 °C sludge. In the acetate conversion study, residual acetate (79 mg l⁻¹) at 70 °C was three to five times higher than that at 55 and 35 °C. Further, the methane produced as percentage of the acetate consumed at 70 °C (89%) was lower than that at 55 (95%) and 35 °C (97%). At 70 °C, 10% of the ISMA remained after 15 days of starvation as compared to 26% (55 °C) and 92% (35 °C) after 30 days of starvation. Thus, the kinetics of the 70 °C granular sludge seem to differ from those at 55 and 35 °C. Received: 1 February 1999 / Accepted: 20 March 1999     

Cavamax W7 composite ethosomal gel of clotrimazole for improved topical delivery: development and comparison with ethosomal gel

The present research work was aimed to formulate clotrimazole encapsulated Cavamax W7 composite ethosomes by injection method for improved delivery across epidermis. 3² factorial design was used to design nine formulations (F1-F9) and compared with ethosomal formulations (F10-F12). F9 with vesicle size of 202.8 ± 4.8 nm, highest zeta potential (−83.6 ± 0.96 mV) and %EE of 98.42 ± 0.15 was selected as optimized composite ethosome and F12 as reference ethosomal formulation. As revealed by transmission electron microscopy F9 vesicles were more condensed, uniformly spherical in shape than F12 vesicles. Vesicular stability studies indicated F9 to be more stable as compared to F12. Both F9 and F12 were incorporated in carbopol 934 gel base to get G1–G8 gel formulations and evaluated for in vitro skin permeability. Cavamax W7 composite ethosomal optimized gel (G5) showed higher in vitro percent cumulative drug permeation (88.53 ± 2.10%) in 8 h and steady state flux (J _ss) of 3.39 ± 1.45 μg/cm²/min against the J _ss of 1.57 ± 0.23 μg/cm²/min for ethosomal gel (G1) and 1.13 ± 0.06 μg/cm²/min for marketed formulation. The J _ss flux of G5 was independent of amount of drug applied/unit area of skin. In vivo confocal laser scanning microscopic study of G5 depicted uniform and deeper penetration of rhodamine B (marker) in epidermis from Cavamax W7 composite ethosomal gel in comparison to G1. Finally, G5 demonstrated better (p < 0.05) antifungal activity against Candida albicans and Aspergillus niger than G1 thus, signifying that Cavamax W7 composite ethosomes present a superior stable and efficacious vesicular system than ethosomal formulation for topical delivery of clotrimazole.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

NMR Reinvestigation of the Caffeine–Chlorogenate Complex in Aqueous Solution and in Coffee Brews

Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate complex in freshly prepared coffee brews have been investigated by high-resolution ¹H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the average value of the self-association constant determined by isodesmic model (K _i = 7.6 ± 0.5 M⁻¹) is in good agreement with the average value (K _a = 10 ± 1.8 M⁻¹) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight decreased tendency to aggregation with a lower average value of association constants (K _i = 2.8 ± 0.6 M⁻¹; K _a = 3.4 ± 0.6 M⁻¹) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex (30 ± 4 M⁻¹) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed. Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine is complexed in beverage real system, being chlorogenate ions the main complexing agents.     

CaATP inhibition of the MgATP-dependent proton pump (H+-ATPase) in bacterial photosynthetic membranes with a mechanism of alternative substrate inhibition

 The membrane-bound F1 sector of the H⁺–ATPase complex (F-type ATPase) in dark-adapted photosynthetic chromatophores is endowed with MgATP- and CaATP-dependent ATPase activities, both sensitive to inhibitors such as oligomycin and venturicidin. Because of contatamination of free Mg^{2 +} and Ca²⁺ ions in chromatophore preparations, kinetic characterization of the two hydrolitic reactions can be performed only in the presence of both substrates, using a model for two alternative substrates. The two activities are characterized by similar maximal rates and affinity constants [V_MgATP and V_CaATP: 13±1 and 10±1 nmol s^–1 ATP hydrolyzed (μmol BChl)^–1; K_MgATP and K_CaATP: 0.22±0.06 and 0.20±0.05 mm]. However, only the MgATP-dependent ATPase is coupled to Δ*_{H +} generation. In this process CaATP acts as an alternative substrate and a competitive inhibitor of the proton pump, with a K_I coincident with K_CaATP for the hydrolytic activity. This finding highlights the central role that the coordination chemistry of the ion-nucleotide complex plays in determining the proton gating mechanism at the catalytic site(s) of the enzyme complex. These results are discussed on the basis of the coordination properties of the ions and of the available information on the protein structure. Received: 5 December 1995 / Accepted: 7 March 1996     

Effect of stress pretreatment on survival of probiotic bacteria in gastrointestinal tract simulator

The effect of stress pretreatment on survival of probiotic Lactobacillus acidophilus La-5, Lactobacillus rhamnosus GG, and Lactobacillus fermentum ME-3 cultures was investigated in the single bioreactor gastrointestinal tract simulator (GITS). The cultures were pregrown in pH-auxostat, subjected to temperature, acid, or bile stress treatment, fast frozen in liquid nitrogen (LN₂), and tested for survival in GITS. After LN₂ freezing the colony forming ability of L. rhamnosus GG and L. fermentum ME-3 nonstressed and stressed cells was well retained (average survival of 75.4 ± 18.3% and 88.0 ± 7.2%, respectively). L. acidophilus La-5 strain showed good survival of auxostat nonstressed cells after fast freezing (94.2 ± 15.0), however the survival of stress pretreated cells was considerably lower (30.8 ± 8.5%). All LN₂ frozen auxostat cultures survived well in the acid phase of the GIT simulation (survival 81 ± 21%); however, after the bile phase, the colony formation ability of L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 decreased by approximately 1.4 ± 0.2, 3.8 ± 0.3, and 3.5 ± 1.2 logarithmic units, respectively. No statistically relevant positive effect of stress pretreatments on survival of LN₂ frozen L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 in GITS was observed.     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ ³²P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [³²P]-inorganic phosphate (³²Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ ³²P]ATP to [³²P]UTP, while inclusion of AMP resulted in conversion of [γ ³²P]ATP to [³²P]ADP. Ebselen markedly reduced [³²P]UTP formation but displayed negligible effect on ³²Pi or [³²P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC₅₀ = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the V _max of the reaction (K _i = 7.6 ± 3 μM), having negligible effect on K _M values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK.     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Characterization of a novel ginsenoside-hydrolyzing α-l-arabinofuranosidase, AbfA, from Rhodanobacter ginsenosidimutans Gsoil 3054T

The gene encoding an α-l-arabinofuranosidase that could biotransform ginsenoside Rc {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-[α-l-arabinofuranosyl-(1–6)-β-d-glucopyranosyl]-20(S)-protopanaxadiol} to ginsenoside Rd {3-O-[β-d-glucopyranosyl-(1–2)-β-d-glucopyranosyl]-20-O-β-d-glucopyranosyl-20(S)-protopanaxadiol} was cloned from a soil bacterium, Rhodanobacter ginsenosidimutans strain Gsoil 3054^T, and the recombinant enzyme was characterized. The enzyme (AbfA) hydrolyzed the arabinofuranosyl moiety from ginsenoside Rc and was classified as a family 51 glycoside hydrolase based on amino acid sequence analysis. Recombinant AbfA expressed in Escherichia coli hydrolyzed non-reducing arabinofuranoside moieties with apparent K _m values of 0.53 ± 0.07 and 0.30 ± 0.07 mM and V _max values of 27.1 ± 1.7 and 49.6 ± 4.1 μmol min⁻¹ mg⁻¹ of protein for p-nitrophenyl-α-l-arabinofuranoside and ginsenoside Rc, respectively. The enzyme exhibited preferential substrate specificity of the exo-type mode of action towards polyarabinosides or oligoarabinosides. AbfA demonstrated substrate-specific activity for the bioconversion of ginsenosides, as it hydrolyzed only arabinofuranoside moieties from ginsenoside Rc and its derivatives, and not other sugar groups. These results are the first report of a glycoside hydrolase family 51 α-l-arabinofuranosidase that can transform ginsenoside Rc to Rd.     

Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate

 Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q _P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K _S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q _S,max, varied between 4.32±0.02 g lactose g cells^-1 h^-1 and 4.89 ±0.02 g lactose g cells^-1 h^-1. The initial specific lactose uptake rate for non-growing cells, q _S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells. Received: 4 October 1995/Received last revision: 23 April 1996/Accepted: 29 April 1996     

Production and properties of a dextransucrase from Leuconostoc mesenteroides IBT-PQ isolated from ‘pulque’, a traditional Aztec alcoholic beverage

Dextransucrase was produced from a Leuconostoc mesenteroides isolated from pulque, a traditional Aztec alcoholic beverage produced from agave juice containing sucrose as the main carbon source. Almost all the dextransucrase activity (87%) was associated with the cells, and was unusually high (1.04 U mg⁻¹ of cells). The culture medium composition was optimized through a Box-Behnken method resulting in a process yielding 2.2 U ml⁻¹ of insoluble glucosyltransferase activity. The enzyme had a molecular weight of 166 kDa. Optimal temperature was 35°C with a half-life of 137 min at the same temperature. As with dextransucrase from the industrial strain L. mesenteroides NRRL B-512F, the enzyme showed Michaelis–Menten kinetic behavior with excess substrate inhibition (K _m and K _i values of 0.026 M and 1.23 M respectively); produced soluble linear dextran with glucose molecules linked mainly in α(1–6) with branching in α(1–3) in a proportion of 4:1 as shown by NMR studies; and produced a high yield of isomalto-oligosaccharides in the presence of maltose. Received 4 February 1998/ Accepted in revised form 25 July 1998     

Bioactive metabolites from <Emphasis Type="Italic">Phoma</Emphasis> species,an endophytic fungus from the Chinese medicinal plant <Emphasis Type="Italic">Arisaema erubescens</Emphasis>

Through bioassay-guided fractionation, the EtOAc extract of a culture broth of the endophytic fungus Phoma species ZJWCF006 in Arisaema erubescens afforded a new α-tetralone derivative, (3S)-3,6,7-trihydroxy-α-tetralone (1), together with cercosporamide (2), β-sitosterol (3), and trichodermin (4). The structures of compounds were established on the basis of spectroscopic analyses. Compounds 1, 2, and 3 were obtained from Phoma species for the first time. Additionally, the compounds were subjected to bioactivity assays, including antimicrobial activity, against four plant pathogenic fungi (Fusarium oxysporium, Rhizoctonia solani, Colletotrichum gloeosporioides, and Magnaporthe oryzae) and two plant pathogenic bacteria (Xanthomonas campestris and Xanthomonas oryzae), as well as in vitro antitumor activities against HT-29, SMMC-772, MCF-7, HL-60, MGC80-3, and P388 cell lines. Compound 1 showed growth inhibition against F. oxysporium and R. solani with EC₅₀ values of 413.22 and 48.5 μg/mL, respectively. Additionally, compound 1 showed no cytotoxicity, whereas compound 2 exhibited cytotoxic activity against the six tumor cell lines tested, with IC₅₀ values of 9.3 ± 2.8, 27.87 ± 1.78, 48.79 ± 2.56, 37.57 ± 1.65, 27.83 ± 0.48, and 30.37 ± 0.28 μM, respectively. We conclude that endophytic Phoma are promising sources of natural bioactive and novel metabolites.     

Antioxidant activity of oxygen-containing aromatic compounds

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H₂O₂-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K _i, μM) or the inhibition degree (%). K _i values varied in the range of 4 to 500 μM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds of guaiacyl and syringal series exhibited high antioxidant activity (K _i in the range of 10–300 μM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycoumaric acid.