A Superoxide Dismutase Purified from the Rhizome of <Emphasis Type="Italic">Curcuma aeruginosa</Emphasis> Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K _m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V _max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min⁻¹ mg protein⁻¹. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn²⁺and Fe²⁺ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC₅₀ = 14.36 ± 0.15 μg protein/ml).     

Cell membrane chromatography competitive binding analysis for characterization of α<Subscript>1A</Subscript> adrenoreceptor binding interactions

A new high α_1A adrenoreceptor (α_1AAR) expression cell membrane chromatography (CMC) method was developed for characterization of α_1AAR binding interactions. HEK293 α_1A cell line, which expresses stably high levels of α_1AAR, was used to prepare the stationary phase in the CMC model. The HEK293 α_1A/CMC-offline-HPLC system was applied to specifically recognize the ligands which interact with the α_1AAR, and the dissociation equilibrium constants (K _D) obtained from the model were (1.87 ± 0.13) × 10⁻⁶ M for tamsulosin, (2.86 ± 0.20) × 10⁻⁶ M for 5-methylurapidil, (3.01 ± 0.19) × 10⁻⁶ M for doxazosin, (3.44 ± 0.19) × 10⁻⁶ M for terazosin, (3.50 ± 0.21) × 10⁻⁶ M for alfuzosin, and (7.57 ± 0.31) × 10⁻⁶ M for phentolamine, respectively. The competitive binding study between tamsulosin and terazosin indicated that the two drugs interacted at the common binding site of α_1AAR. However, that was not the case between tamsulosin and oxymetazoline. The results had a positive correlation with those from radioligand binding assay and indicated that the CMC method combined modified competitive binding could be a quick and efficient way for characterizing the drug–receptor interactions.     

Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis

Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-d-glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC ₅₀ ) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease.     

Angiotensin I-Converting Enzyme Inhibitory Proteins and Peptides from the Rhizomes of Zingiberaceae Plants

Ammonium sulphate cut protein extracts, and their pepsin hydrolysates, from the rhizomes of 15 plants in the Zingiberaceae family were screened for their in vitro angiotensin I-converting enzyme inhibitory (ACEI) activity. The protein extract from Zingiber ottensii had the highest ACEI activity (IC₅₀ of 7.30 × 10⁻⁷ mg protein/mL) and was enriched for by SP Sepharose chromatography with five NaCl step gradients 0, 0.25, 0.50, 0.75 and 1 M NaCl collecting the corresponding five fractions. The highest ACEI activity was found in the F75 fraction, which appeared to contain a single 20.7-kDa protein, suggesting enrichment to or near to homogeneity. The ACEI activity of the F75 fraction was moderately thermostable (−20–60 °C), showed >80% activity across a broad pH range of 4–12 (optimal at pH 4–5) and appeared as a competitive inhibitor of ACE (K _i of 9.1 × 10⁻⁵ mg protein/mL). For the pepsin hydrolysates, that from Zingiber cassumunar revealed the highest ACEI activity (IC₅₀ of 0.38 ± 0.012 mg/mL), was enriched to a single active hexapeptide by RP-HPLC with a strong ACEI activity (IC₅₀ of 0.011 ± 0.012 mg/mL) and acted as a competitive inhibitor of ACE (K _i of 1.25 × 10⁻⁶ mg protein/mL).     

Interaction of partially ionized poly(acrylic acid) with a surfactant counterion at constant pH values

Binding of a cationic surfactant ion, dodecylpyridinium ion, to poly(acrylic acids) of low charge densities was examined by potentiometry using surfactant-selective electrodes in the solutions, where the pH was kept constant by employing a pH buffering system. The binding of the surfactant counterions was thus able to be studied at a constant pH during the binding process. The binding took place in two steps, the first cooperative binding step and the second gradual binding step. The critical association concentration decreased as the pH increased, indicating the predominant role of the electric interaction in the binding. The binding isotherms obtained at different but constant pH values were analyzed by the matrix method, taking into account the nearest-neighbor interactions among three different kinds of sites on the polymer: ionized, protonated, and surfactant-bound. The theoretical analysis could describe only the first step but could not explain the second step. A relatively large cooperativity parameter, u, was found for the first step and it can be between 3 × 10³ and 1 × 10⁴. When the ionic strength was decreased tenfold, the cooperativity of the binding decreased (u∼1 × 10³). The binding constants of the isolated site were 5.5–6.0 × 10⁴ kg mol⁻¹ and slightly increased to 6.5 × 10⁴ kg mol⁻¹ as the ionic strength decreased. The deviation of the second step from the theoretical analysis was supposed to arise from a change of proton dissociation constant in the nonpolar space formed by the bound surfactants. Received: 29 November 2000/Accepted: 24 January 2001     

Complexation of M–(buffer) x –(OH) y Systems Involving Divalent Ions (Cobalt or Nickel) and Zwitterionic Biological Buffers (AMPSO, DIPSO, TAPS and TAPSO) in Aqueous Solution

The complexation behavior of eight M–(buffer) _x –(OH) _y systems involving two divalent ions (cobalt and nickel) and four zwitterionic biological buffers (AMPSO, DIPSO, TAPS and TAPSO) were characterized. Complex formation was detected for all eight M–(buffer) _x –(OH) _y systems studied, but fully defined final models were obtained for only four of these systems. For systems involving cobalt or nickel with AMPSO or TAPS, a complete characterization of the systems was not possible in the studied buffer pH-range. Metal complexation was studied by glass-electrode potentiometry (GEP) and UV-Vis spectroscopy at 25.0 °C and I=0.1 mol⋅dm⁻³ KNO₃ ionic strength. For the Ni–(L) _x –(OH) _y and Co–(L) _x –(OH) _y systems, with L = TAPSO or DIPSO, the proposed final models and overall stability constants were obtained by combining results from both techniques. For the Ni–(L) _x –(OH) _y systems, the measured values of the stability constants are log ₁₀ β _NiL=3.0±0.1 and log ₁₀ β _NiL2=4.8±0.1 for L = TAPSO, and log ₁₀ β _NiL=2.7±0.1 and log ₁₀ β _NiL2=4.6±0.1 for L = DIPSO. For the Co–(L) _x –(OH) _y systems, the overall stability constants are log ₁₀ β _CoL=2.2±0.1, log ₁₀ β _CoL2=3.6±0.2 and log ₁₀ β _CoL(OH)=7.6±0.1 for L = TAPSO, and log ₁₀ β _CoL=2.0±0.1 and log ₁₀ β _CoL(OH)=7.8±0.1 for L = DIPSO. For both buffers, the CoL(OH) species is characterized by a major structural rearrangement.     

New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen

In the present study, a new fluorescence microplate screening assay for evaluating scavenging activity against singlet oxygen (¹O₂) was implemented. The chemical generation of ¹O₂ was promoted using the thermodissociable endoperoxide of disodium 3,3′-(1,4-naphthalene)bispropionate (NDPO₂). The detection of ¹O₂ was achieved using dihydrorhodamine 123 (DHR), a nonfluorescent molecule that is oxidizable to the fluorescent form rhodamine 123 (RH). The combined use of a ¹O₂-selective generator and a highly sensitive probe (DHR) was then successfully applied to perform a screening assay of the ¹O₂ scavenging activities of ascorbic acid, penicillamine, cysteine, N-acetylcysteine (NAC), methionine, reduced glutathione (GSH), dihydrolipoic acid, lipoic acid, and sodium azide. All of these antioxidants exhibited concentration-dependent ¹O₂ scavenging capacities. They could be ranked according to observed activity: ascorbic acid> cysteine> penicillamine> dihydrolipoic acid > GSH> NAC> sodium azide> lipoic acid (IC₅₀ values of 3.0 ± 0.2, 8.0 ± 0.7, 10.9 ± 0.8, 25.2 ± 4.5, 57.4 ± 5.9, 138 ± 13, 1124 ± 128, 2775 ± 359 μM, mean±SEM, respectively) > methionine (35% of scavenging effect at 10 mM). In conclusion, the use of NDPO₂ as a selective generator for ¹O₂ and its fluorescence detection by the highly sensitive probe DHR is shown to be a reliable and resourceful analytical alternative means to implement a microplate screening assay for scavenging activity against ¹O₂. Generation and detection of singlet oxygen     

Simultaneous determination of harpagoside and cinnamic acid in rat plasma by high-performance liquid chromatography: application to a pharmacokinetic study

Radix Scrophulariae (Xuanshen) is one of the famous Chinese herbal medicines widely used to treat rheumatism, tussis, pharyngalgia, arthritis, constipation, and conjunctival congestion. Harpagoside and cinnamic acid are the main bioactive components of Xuanshen. The purpose of this study was to develop an HPLC–UV method for simultaneous determination of harpagoside and cinnamic acid in rat plasma and investigate pharmacokinetic parameters of harpagoside and cinnamic acid after oral administration of Xuanshen extract (760 mg kg⁻¹). After addition of syringin as internal standard, the analytes were isolated from plasma by liquid–liquid extraction. Separation was achieved on a Kromasil C₁₈ column, and detection was by UV absorption at 272 nm. The described assay was validated in terms of linearity, accuracy, precision, recovery, and limit of quantification according to the FDA validation guidelines. Calibration curves for both analytes were linear with the coefficient of variation (r) for both was greater than 0.999. Accuracy for harpagoside and cinnamic acid ranged from 100.7–103.5% and 96.9–102.9%, respectively, and precision for both analytes were less than 8.5%. The main pharmacokinetic parameters found for harpagoside and cinnamic acid after oral infusion of Xuanshen extract were as follows: C _max 1488.7 ± 205.9 and 556.8 ± 94.2 ng mL⁻¹, T _max 2.09 ± 0.31 and (1.48 ± 0.14 h, AUC_0–24 10336.4 ± 1426.8 and 3653.1 ± 456.4 ng h mL⁻¹, 11276.8 ± 1321.4 and 3704.5 ± 398.8 ng h mL⁻¹, and t _1/2 4.9 ± 1.3 and 2.5 ± 0.9 h, respectively. These results indicated that the proposed method is simple, selective, and feasible for pharmacokinetic study of Radix Scrophulariae extract in rats. Figure Radix Scrophulariae     

Identification and analysis of stereoselective drug interactions with low-density lipoprotein by high-performance affinity chromatography

Columns containing immobilized low-density lipoprotein (LDL) were prepared for the analysis of drug interactions with this agent by high-performance affinity chromatography (HPAC). R/S-Propranolol was used as a model drug for this study. The LDL columns gave reproducible binding to propranolol over 60 h of continuous use in the presence of pH 7.4 0.067 M potassium phosphate buffer. Experiments conducted with this type of column through frontal analysis indicated that two types of interactions were occurring between R-propranolol and LDL, while only a single type of interaction was observed between S-propranolol and LDL. The first type of interaction, which was seen for both enantiomers, involved non-saturable binding; this interaction had an overall affinity (nK _a) of 1.9 (±0.1) × 10⁵ M⁻¹ for R-propranolol and 2.7 (±0.2) × 10⁵ M⁻¹ for S-propranolol at 37 °C. The second type of interaction was observed only for R-propranolol and involved saturable binding that had an association equilibrium constant (K _a) of 5.2 (±2.3) × 10⁵ M⁻¹ at 37 °C. Similar differences in binding behavior were found for the two enantiomers at 20 °C and 27 °C. This is the first known example of stereoselective binding of drugs by LDL or other lipoproteins. This work also illustrates the ability of HPAC to be used as a tool for characterizing mixed-mode interactions that involve LDL and related binding agents.     

The Correlations Between TCA-Glyoxalate Metabolite and Antibiotic Production of <Emphasis Type="Italic">Streptomyces</Emphasis> sp. M4018 Grown in Glycerol,Glucose, and Starch Mediums

The alterations of organic acids citrate, α-ketoglutarate, succinate, fumarate, malate production together with isocitrate lyase activity as a glyoxalate shunt enzyme, and antibiotic production of Streptomyces sp M4018 were investigated in relation to changes in the glucose, glycerol and starch concentrations (5–20 g/L) after identification as a strain of Streptomyces hiroshimensis based on phenotypic and genotypic characteristics. The highest intracellular citrate and α-ketoglutarate levels in 20 g/l of glucose, glycerol, and starch mediums were 399.47 ± 4.78, 426.93 ± 6.40, 355.84 ± 5.38 ppm and 444.81 ± 5.12, 192.96 ± 2.26, 115.20 ± 2.87 ppm, respectively. The highest succinate, malate, and fumarate levels were also determined in 20 g/l of glucose medium as 548.9 ± 11.21, 596.15 ± 8.26, and 406.42 ± 6.59 ppm and the levels were significantly higher than the levels in glycerol and starch. Extracellular organic acid levels measured also showed significant correlation with carbon source concentrations by showing negative correlation with pH levels of the growth medium. The antibiotic production of Streptomyces sp. M4018 was also higher in glucose medium as was the case also for organic acids when compared with glycerol. On the other hand, there is no production in starch.     

Contribution of Glycerol on Production of Poly(γ-Glutamic Acid) in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> NX-2

Glycerol would stimulate the production of poly(γ-glutamic acid) (γ-PGA) and decrease its molecular weight in Bacillus subtilis NX-2. When 20 g/l glycerol was added in the medium, the yield of γ-PGA increased from 26.7 ± 1.0 to 31.7 ± 1.3 g/l, and molecular weight of γ-PGA decreased from 2.43 ± 0.07 × 10⁶ to 1.86 ± 0.06 × 10⁶ Da. In addition, it was found that the decrease of γ-PGA chain length by glycerol would lead to the decrease of broth viscosity during the fermentation and enhanced the uptake of substrates, which could not only improve cell growth but also stimulate γ-PGA production. Moreover, it was also found that glycerol could effectively regulate molecular weight between 2.43 ± 0.07 × 10⁶ and 1.42 ± 0.05 × 10⁶ Da with the concentration ranging from 0 to 60 g/l. This was the first time to discover such contribution of glycerol on γ-PGA production in Bacillus genus. And the effects of glycerol on molecular weight of γ-PGA would be developed to be an approach for the regulation of microbial γ-PGA chain length, which is of practical importance for future commercial development of this polymer.     

Characterization of currently marketed heparin products: analysis of molecular weight and heparinase-I digest patterns

We evaluated polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) approaches to determine weight-average molecular weight (M _w) and polydispersity (PD) of heparins. A set of unfractionated heparin sodium (UFH) and low-molecular-weight heparin (LMWH) samples obtained from nine manufacturers which supply the US market were assessed. For SEC-MALLS, we measured values for water content, refractive index increment (dn/dc), and the second virial coefficient (A ₂) for each sample prior to molecular weight assessment. For UFH, a mean ± standard deviation value for M _w of 16,773 ± 797 was observed with a range of 15,620 to 18,363 (n = 20, run in triplicate). For LMWHs by SEC-MALLS, we measured mean M _w values for dalteparin, tinzaparin, and enoxaparin of 6,717 ± 71 (n = 4), 6,670 ± 417 (n = 3), and 3,959 ± 145 (n = 3), respectively. PAGE analysis of the same UFH, dalteparin, tinzaparin, and enoxaparin samples showed values of 16,135 ± 643 (n = 20), 5,845 ± 45 (n = 4), 6,049 ± 95 (n = 3), and 4,772 ± 69 (n = 3), respectively. These orthogonal measurements are the first M _w results obtained with a large heparin sample set on product being marketed after the heparin crisis of 2008 changed the level of scrutiny of this drug class. In this study, we compare our new data set to samples analyzed over 10 years earlier. In addition, we found that the PAGE analysis of heparinase digested UFH and neat LMWH samples yield characteristic patterns that provide a facile approach for identification and assessment of drug quality and uniformity.     

α-Mannosidases from the Digestive Fluid of <Emphasis Type="Italic">Rhynchophorus Palmarum</Emphasis> Larvae as Novel Biocatalysts for Transmannosylation Reactions

Two biological fluids, namely hemolymph and digestive fluid from the larval stage of Rhynchophorus palmarum Linneaus, a serious pest in agroecosystem exploiting oil palm, were screened for hydrolytic activities, by the use of synthetic and natural glycoside substrates. Several exo and endoglycosidase activities were observed but, the interesting α-mannosidase activity (0.41 ± 0.04 UI) had attracted our attention. So, we have previously demonstrated that this activity harbours four distinctive α-mannosidase isoforms named RpltM, RplM1, RplM2 and RplM3. We have extended this work to determine the ability of these enzymes to catalyze synthesis reactions. Finally, we have revealed that, α-mannosidases from the digestive fluid of R. palmarum larvae catalyze transmannosylation reactions. The stability of the enzymes and the optimization of the transfer product yield were studied as functions of pH, enzyme unit, starting concentration of donor or acceptor and time. It was shown that, in experimental optimum conditions, average yields of 12.34 ± 0.75, 12.15 ± 0.79, 5.59 ± 0.35 and 8.43 ± 0.50% were obtained for the α-mannosidases RpltM, RplM1, RplM2 and RplM3, respectively. On the basis of this work, α-mannosidases from the digestive fluid of Rhynchophorus palmarum larvae appear to be a valuable tool for the preparation of neoglycoconjugates.     

An Improved Enzyme Assay for Molybdenum-Reducing Activity in Bacteria

Molybdenum-reducing activity in the heterotrophic bacteria is a phenomenon that has been reported for more than 100 years. In the presence of molybdenum in the growth media, bacterial colonies turn to blue. The enzyme(s) responsible for the reduction of molybdenum to molybdenum blue in these bacteria has never been purified. In our quest to purify the molybdenum-reducing enzyme, we have devised a better substrate for the enzyme activity using laboratory-prepared phosphomolybdate instead of the commercial 12-phosphomolybdate we developed previously. Using laboratory-prepared phosphomolybdate, the highest activity is given by 10:4-phosphomolybdate. The apparent Michaelis constant, K _m for the laboratory-prepared 10:4-phosphomolybdate is 2.56 ± 0.25 mM (arbitrary concentration), whereas the apparent V _max is 99.4 ± 2.85 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent Michaelis constant or K _m for NADH as the electron donor is 1.38 ± 0.09 mM, whereas the apparent V _max is 102.6 ± 1.73 nmol Mo-blue min⁻¹ mg⁻¹ protein. The apparent K _m and V _max for another electron donor, NADPH, is 1.43 ± 0.10 mM and 57.16 ± 1.01 nmol Mo-blue min⁻¹ mg⁻¹ protein, respectively, using the same batch of molybdenum-reducing enzyme. The apparent V _max obtained for NADH and 10:4-phosphomolybdate is approximately 13 times better than 12-phoshomolybdate using the same batch of enzyme, and hence, the laboratory-prepared phosphomolybdate is a much better substrate than 12-phoshomolybdate. In addition, 10:4-phosphomolybdate can be routinely prepared from phosphate and molybdate, two common chemicals in the laboratory.     

High Level Expression of an Acid-Stable Phytase from <Emphasis Type="Italic">Citrobacter freundii</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni²⁺–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg⁻¹, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg⁻¹ min⁻¹, respectively. The enzyme activity was significantly improved by 1 mM of K⁺, Ca²⁺, and Mg²⁺. These characteristics contribute to its potential application in feed industry.     

Colloidal crystals made of poly(N-isopropylacrylamide) microgel particles

Poly (N-isopropylacrylamide) microgel particles are found to form colloidal crystals similar to those occurring in typical hard-sphere colloids like poly(methylmethacrylate) beads. Samples made of particles with different cross-linker concentrations are investigated and their deswelling ratio is determined using dynamic light scattering. Small-angle neutron scattering data are also presented and analysed in terms of a face-centred-cubic crystal structure. The characteristic length, a, of the elementary cell is found to be 535 ± 16 and 495 ± 15 nm for the two systems investigated. This leads to particle radii of 189 ± 6 and 175 ± 5 nm, respectively. These values compare well to the radii determined using several different methods. Received: 26 July 1999/Accepted: 21 March 2000     

Determination of enzyme activity inhibition by FTIR spectroscopy on the example of fructose bisphosphatase

A mid-infrared enzymatic assay for label-free monitoring of the enzymatic reaction of fructose-1,6-bisphosphatase with fructose 1,6-bisphosphate has been proposed. The whole procedure was done in an automated way operating in the stopped flow mode by incorporating a temperature-controlled flow cell in a sequential injection manifold. Fourier transform infrared difference spectra were evaluated for kinetic parameters, like the Michaelis–Menten constant (K _M) of the enzyme and V _max of the reaction. The obtained K _M of the reaction was 14 ± 3 g L⁻¹ (41 μM). Furthermore, inhibition by adenosine 5′-monophosphate (AMP) was evaluated, and the K _M^App value was determined to be 12 ± 2 g L⁻¹ (35 μM) for 7.5 and 15 μM AMP, respectively, with V _max decreasing from 0.1 ± 0.03 to 0.05 ± 0.01 g L⁻¹ min⁻¹. Therefore, AMP exerted a non-competitive inhibition.     

Binding properties of a monoclonal antibody against the Cry1Ab from <Emphasis Type="Italic">Bacillus Thuringensis</Emphasis> for the development of a capillary electrophoresis competitive immunoassay

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 10⁶ M⁻¹ and (9.06 ± 5.7) × 10⁶ M⁻¹—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L⁻¹). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.     

Investigation of calcium antagonist–L-type calcium channel interactions by a vascular smooth muscle cell membrane chromatography method

The dissociation equilibrium constant (K _D) is an important affinity parameter for studying drug–receptor interactions. A vascular smooth muscle (VSM) cell membrane chromatography (CMC) method was developed for determination of the K _D values for calcium antagonist–L-type calcium channel (L-CC) interactions. VSM cells, by means of primary culture with rat thoracic aortas, were used for preparation of the cell membrane stationary phase in the VSM/CMC model. All measurements were performed with spectrophotometric detection (237 nm) at 37 °C. The K _D values obtained using frontal analysis were 3.36 × 10⁻⁶ M for nifedipine, 1.34 × 10⁻⁶ M for nimodipine, 6.83 × 10⁻⁷ M for nitrendipine, 1.23 × 10⁻⁷ M for nicardipine, 1.09 × 10⁻⁷ M for amlodipine, and 8.51 × 10⁻⁸ M for verapamil. This affinity rank order obtained from the VSM/CMC method had a strong positive correlation with that obtained from radioligand binding assay. The location of the binding region was examined by displacement experiments using nitrendipine as a mobile-phase additive. It was found that verapamil occupied a class of binding sites on L-CCs different from those occupied by nitrendipine. In addition, nicardipine, amlodipine, and nitrendipine had direct competition at a single common binding site. The studies showed that CMC can be applied to the investigation of drug–receptor interactions.     

Biomolecular profiling of metastatic prostate cancer cells in bone marrow tissue using FTIR microspectroscopy: a pilot study

Prostate cancer (CaP) cells preferentially metastasise to the bone marrow, a microenvironment that plays a substantial role in the sustenance and progression of the CaP tumour. Here we use a combination of FTIR microspectroscopy and histological stains to increase molecular specificity and probe the biochemistry of metastatic CaP cells in bone marrow tissue derived from a limited source of paraffin-embedded biopsies of different patients. This provides distinction between the following dominant metabolic processes driving the proliferation of the metastatic cells in each of these biopsies: glycerophospholipid synthesis from triacylglyceride, available from surrounding adipocytes, in specimen 1, through significantly high (p ≤ 0.05) carbohydrate (8.23 ± 1.44 cm⁻¹), phosphate (6.13 ± 1.5 cm⁻¹) and lipid hydrocarbon (24.14 ± 5.9 cm⁻¹) signals compared with the organ-confined CaP control (OC CaP), together with vacuolation of cell cytoplasm; glycolipid synthesis in specimen 2, through significantly high (p ≤ 0.05) carbohydrate (5.51 ± 0.04 cm⁻¹) and high lipid hydrocarbon (17.91 ± 2.3 cm⁻¹) compared with OC CaP, together with positive diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells; glycolysis in specimen 3, though significantly high (p ≤ 0.05) carbohydrate (8.86 ± 1.78 cm⁻¹) and significantly lower (p ≤ 0.05) lipid hydrocarbon (11.67 ± 0.4 cm⁻¹) than OC CaP, together with negative diastase-digested periodic acid Schiff staining in the majority of metastatic CaP cells. Detailed understanding of the biochemistry underpinning the proliferation of tumour cells at metastatic sites may help towards refining chemotherapeutic treatment.