Possibilitiés d'étude des variantes de la cholinestérase plasmatique humaine par électrophorèse d'affinitéPossibilities of study of the human plasma cholinesterase variants by affinity electrophoresis

Affinity electrophoresis has been applied to the study of the multiple molecular forms of three human plasma cholinesterase phenotypes (usual enzyme U, atypical enzyme A and intermediate UA). Electrophoreses were carried out in polyacrylamide gels containing a water-soluble macromolecular derivative of m-amino-(substituted)-phenyltrimethylammonium immobilized within the gel network.Apparent dissociation constants (K_{D app}) were estimated from the mobilities of the enzymes versus ligand concentration.The ratio of K_{D app} values of the molecular forms of phenotypes A and U which is approximately 2 is consistent with the hypothesis that the anionic site is altered in atypical enzyme.     

The titratable joint phenomenon in ΦW-14 DNA

Dynamic light scattering (DLS) studies are carried out on native ΦW-14 DNA, which has putrescines covalently attached at the methyl groups of half its thymines, and on a chemically modified form of the same DNA in which the ammonium groups of its putrescines are almost completely acetylated. From neutrality to pH 9.6, both forms of ΦW-14 DNA exhibit the same curve of D_app vs K² over the range K² = 0.5 × 10¹⁰ to K² = 20 × 10¹⁰ cm^?2, and this coincides with curves that we have observed for other DNAs. (D_app, apparent diffusion coefficient; K, scattering vector). However, when the pH is raised to pH 10.0–10.2, native ΦW-14 exhibits a spectacular decrease in D_app at large wave vector, whereas the acetylated form shows no sign of such behavior. It is inferred that bound ammonium groups make an essential contribution to the stabilization of titratable joints. Comparing the pH profiles of the absorbance (A₂₆₀) for these two DNAs gives some evidence that base unstacking may be involved in titratable joint formation.     

Boundaries of the universal K3 region and plateau region of the dynamic structure factor for DNA

A sufficiently long semiflexible filamentous macromolecule is theroretically expected to exhibit three different domains of behavior of its apparent diffusion coefficient D_app(K) as a function of scattering vector K: (1) the small wave vector limit, where D_app(K) = D₀ is the translational diffusion coefficient of the center-of-mass; (2) the universal K³ region, where D_app(K) = (k_BT/6πη)K is a universal function of K independent of any property of the molecule itself; (c) the plateau region at large K², where D_app(K) approaches either a plateau, or gradually sloping quasiplateau, characteristic of local (elastic) rigid-body motions of the filament. The existence of each of these different domains has now been established experimentally for at least some polymers. The boundaries of the universal K³ region and the plateau region are determined theoretically here using precise quantitative criteria for universal or plateau behavior of D_app(K) for a Rouse-Zimm model containing N + 1 subchains with rms subchain extension b. Allowing a maximum of 13% nonuniversal behavior, the domain of the universal K³ region is given by K²R²_G = K²Nb²/6 ≥ 7 and K²b² ? 0.54. Allowing as much as 10% nonplateau behavior, the boundary for onset of plateau behavior is K²b² = 18.3. D_app(K) is at least 50% nonuniversal when K²b²/6 = 6 ln 3. Extension of these results to DNA is examined theoretically, and good agreement of the pertinent predictions with published experimental data is demonstrated. It is concluded that no truly universal K³ region exists for DNA with M_r ? 10⁷ and persistence length a ≥ 450 Å, although marginally (?17% nonuniversal) universal behavior, is exhibited in a very narrow domain 0.64 × 10¹⁰ ? K² ? 0.84 × 10¹⁰ cm^?2 for ?29 DNA (M_r = 11.5 × 10⁶). More than 50% of D_app(K) is governed by local (elastic) rigid-body motions when K² = 5.23 × 10¹⁰ cm^?2. The existence of a very wide region of nonuniversal apparent K³ behavior extending up to very large K², far into the plateau region, is demonstrated in a plot of D_app(K)/K vs K² for the Rouse-Zimm model. This is shown to stem in part from visual artifacts of plotting D_app(K)/K vs K², even for rigid species.     

Genetically defined peptidases of maize I. Biochemical characterization of allelic and nonallelic forms

A number of biochemical properties have been investigated for both allelic and nonallelic forms of maize peptidases. Four aminopeptidases exist in maize (LAP-A, LAP-B, LAP-C, and LAP-D) and are the products of four diallelic loci. The aminopeptidases fall into two biochemical groups on the basis of these studies. LAP-A and LAP-D have comparatively low apparent K _m (K _app) values for arginine-naphthylamide derivatives and high velocities for arginine-naphthylamide and lysine-naphthylamide. LAP-B and LAP-C, on the other hand, have lower K _app values for leucine-naphthylamide and higher velocities for nonpolar amino acid-naphthylamides than for arginine-naphthylamide. LAP-A and LAP-D are also relatively more heat stable than LAP-B and LAP-C and have somewhat higher molecular weights (71,500) than LAP-B and LAP-C (63,500). In determining molecular weights of the peptidases, use was made of their differential substrate specificities toward amino acid-naphthylamides. Some properties of genetically defined maize endopeptidase are also presented. Maize endopeptidase is inhibited by the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate (pCMB), and by tosyl lysine chloromethyl ketone. Maize aminopeptidase activity is inhibited by N-ethylmaleimide, pCMB, and EDTA (ethylenediamine tetraacetic acid).This research was supported by U.S. Atomic Energy Commission Contract AT(38-1)-770, and in part by Grant No. GM-22733 from the National Institute of General Medical Sciences, U.S. Public Health Service, to J. G. S.Paper No. 4740 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Dynamic light-scattering studies of internal motions in DNA. I. Applicability of the rouse-zimm model

The intermediate scattering function G(K,t) for any polymer model obeying a linear separable Langevin equation can be expressed in terms of the eigenvalues and eigenvectors of its normal coordinate transformation. An algorithm for the extract numerical evaluation of G(K,t) for linear Rouse-Zimm chains in the presence of hydrodynamic interaction has been developed. The computed G(K,t)² were fit to C(t) = A exp(?t/τA) + B, and apparent diffusion coefficients calculated according to D_app ≡ 1/(2τ_AK²). G(K,t)² was surprisingly well-fit by single-exponential decays, especially at both small and large values of Kb, where K is the scattering vector and b the root-mean-squared subunit extension. Plots of D_app vs K² in-variably showed a sigmoidal rise from D₀ at K² = O up to a constant plateau value at large K²b². Analytical expression for G(K,t), exact in the limit of short times, were obtained for circular Rouse-Zimm chains with and without hydrodynamic interaction, and also for free-draining linear chains, and in addition for the independent-segment-mean-force (ISMF) model. The predicted behaviors for G(K,t) at large Kb (or KR_G) was found in all cases to be single-exponential with 1/τ ∝ K² at large Kb, in agreement with the computational results. A simple procedure for estamating all parameter of the Rouse-Zimm model from a plot of D_app vs K² is proposed. Experimental data for both native and pH-denatured calf-thymus DNA in 1.0M Nacl with and without EDTA clearly plateau behavior of D_app at large values of K, in harmony with the present Rouse-Zimm and ISMF theories, and in sharp contrast to previous predictions based on the Rouse-Zimm model.     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.     

Cytochrome P450 125 (CYP125) catalyses C26‐hydroxylation to initiate sterol side‐chain degradation in Rhodococcus jostii RHA1

The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3‐hydroxysterols such as cholesterol, but not on 3‐oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side‐chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side‐chain. The RG32Ωcyp125 mutant failed to transform the side‐chain of cholesterol, but degraded that of 5‐cholestene‐26‐oic acid‐3β‐ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125_RHA1 was < 10% in the low‐spin state, cholesterol (K_D^app = 0.20 ± 0.08 μM), 5α‐cholestanol (K_D^app = 0.15 ± 0.03 μM) and 4‐cholestene‐3‐one (K_D^app = 0.20 ± 0.03 μM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26‐carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.     

Photoaptameric heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinks with a target protein

Using DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nucleotides (nt) in length) to produce aptamer heterodimeric constructs results into affinity enhancement. At the linker lengths ranged from 35 to 55 nt the apparent dissociation constants (K _D^app) measured using the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (K _D^app) = 0.2–0.4 nM), which were approximately 30-fold less than for the complexes with the initial aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer could bind to the exosite 1. The (K _D^app value for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure of the equimolar mixtures of thrombin with the photoaptamer construct to the UV radiation at 308 nm the equal yield of the crosslinked complexes was observed at concentrations, which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.     

Purification and characterization of a thermoalkalophilic xylanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Summary An extracellular xylanase was purified to homogeneity from the culture filtrate of a thermophilic Bacillus sp. The molecular weight of the purified xylanase was 44 kDa, as analysed by SDS/PAGE. The enzyme reaction followed Michaelis–Menten kinetics with K_m^app and V_max values of 0.025 mg/ml and 450 U/mg protein, respectively, as obtained from a Lineweaver–Burk plot. The xylanase contained no other enzyme activity except for the hydrolysis of xylan substrate. The optimal temperature of the enzyme assay was 50 °C. The optimum pH for the xylanase activity was at three peaks 6.5, 8.5 and 10.5, respectively and the enzyme was stable over a broad range of pH from pH 6 to 10.5. Metal ions tested with demetalized enzyme had no effect, with the exception of Hg²⁺ and Pb²⁺ (both strong inhibitors). Inhibition of the enzyme activity by N-bromosuccinimide (amino acid modifier) indicated the role of tryptophan residues in the catalytic function of the enzyme. Due to these outstanding properties, the xylanase of Bacillussp. finds potential applications in biopulping, biobleaching and de-inking of recycled paper and other industrial processes.     

Dynamic light scattering from thin rigid rods: Anisotropy of translational diffusion of tobacco mosaic virus

An Exact theoretical expression for the apparent diffusion coefficient D_app(K) of a thin rigid rod with arbitrary anisotropy of its translational diffusion diffusion coefficient is derived from the first cumulant of its dynamic structure factor. D_app(K) is predicted to reach a limiting plateau value at extermely large values of KL, where K is the scattering vector and L the rod length. Howerver, that limiting plateau value is approached only very slowly along a quasi-plateau with a very gradual slope. Dynamic light-scattering studies have been performed on tobacco mosaic virus from K² = (0.4–20) × 10¹⁰ cm^?2 using 632-8-nm laser radiation. The present data yield D₀ = (4.19 ± 0.10) × 10^?8 cm²/s (corrected to 20,w conditions) and, with literature data to establish L = 2980 Å and the rotational diffusion coefficient D_R = 318s^?1, yield also Δ ≡ D_∥ ? D_⊥ = (1.79 ± 0.38) × 10^?8 cm²/s. The experimental data closely follow the curve of D_app(K) vs K² calcuated for these parameters. The present value of D₀ substantially exceeds all previous dynamic light-scattering values, but is in good aggreement with previous sedimentation data, which were confirmed for the presemt sample. The anisotropy ratio Δ/D₀ = 0.43 ± 0.09 is in accord with theoretical predictions based on the modified Kirkwood algorithm, despite the fact the D₀ lies significantly below its corresponding theoretical value. The present data largely predlude the possibility that both D₀ and Δ/D₀ could simultaneously match their theoretical predictions. We present a detailed comparison of the experimental data with the calculations of Tirado and Garcia de la Torre based on the modified Kirkwood algorithm and with the Broersma formulas.     

Probing a functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase through transesterification reactions in organic solvent

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theV _max,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheK _m,app for tributyrin was not affected by the Glu87Ala mutation, but theK _m,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedV _max,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theK _m,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theK _m,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theV _max,app was only 6% of that obtained with wild-type enzyme.     

Physicochemical properties of hydroxylated polychlorinated biphenyls aid in predicting their interactions with rat sulfotransferase 1A1 (rSULT1A1)

Hydroxylated metabolites of polychlorinated biphenyls (OHPCBs) interact with rat sulfotransferase 1A1 (rSULT1A1) as substrates and inhibitors. Previous studies have shown that there are complex and incompletely understood structure–activity relationships governing the interaction of rSULT1A1 with these molecules. Furthermore, modification of the enzyme with glutathione disulfide (GSSG) results in the conversion of some OHPCBs from inhibitors to substrates. We have now examined estimated values for the acid-dissociation constant (K_a) and the octanol–water distribution coefficient (D), as well as experimentally determined dissociation constants for enzyme complexes, to assist in the prediction of interactions of OHPCBs with rSULT1A1. Under reducing conditions, initial velocities for rSULT1A1-catalyzed sulfation exhibited a positive correlation with pK_a and a negative correlation with log D of the OHPCBs. IC₅₀ values of inhibitory OHPCBs decreased with decreasing pK_a values for both the glutathione (GSH)-pretreated and GSSG-pretreated forms of rSULT1A1. Comparison of GSH- and GSSG-pretreated forms of rSULT1A1 with respect to binding of OHPCB in the presence and absence of adenosine 3′,5′-diphosphate (PAP) revealed that the dissociation constants with the two redox states of the enzyme were similar for each OHPCB. Thus, pK_a and log D values are useful in predicting the binding of OHPCBs to the two redox forms of rSULT1A1 as well as the rates of sulfation of those OHPCBs that are substrates. However, the differences in substrate specificity for OHPCBs that are seen with changes in redox status of the enzyme are not directly related to specific structural effects of individual OHPCBs within inhibitory enzyme–PAP–OHPCB complexes.     

A comparative study of activity and apparent inhibition of fungal β‐glucosidases

β‐Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non‐linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k_cat, K_m, and the apparent product inhibition constant _appK_i for each of the enzymes. A more simple fit is not advisable to use as the determined _appK_i are in the same range as their K_m for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k_cat was determined for A. fumigatus (768 s^?1) and the lowest was a factor 9 less. K_m varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured _appK_i varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of _appK_i using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high. Biotechnol. Bioeng. 2010;107: 943–952. © 2010 Wiley Periodicals, Inc.     

A dominant‐negative form of Arabidopsis AP‐3 β‐adaptin improves intracellular pH homeostasis

Intracellular pH (pH_i) is a crucial parameter in cellular physiology but its mechanisms of homeostasis are only partially understood. To uncover novel roles and participants of the pH_i regulatory system, we have screened an Arabidopsis mutant collection for resistance of seed germination to intracellular acidification induced by weak organic acids (acetic, propionic, sorbic). The phenotypes of one identified mutant, weak acid‐tolerant 1‐1D (wat1‐1D) are due to the expression of a truncated form of AP‐3 β‐adaptin (encoded by the PAT2 gene) that behaves as a as dominant‐negative. During acetic acid treatment the root epidermal cells of the mutant maintain a higher pH_i and a more depolarized plasma membrane electrical potential than wild‐type cells. Additional phenotypes of wat1‐1D roots include increased rates of acetate efflux, K⁺ uptake and H⁺ efflux, the latter reflecting the in vivo activity of the plasma membrane H⁺‐ATPase. The in vitro activity of the enzyme was not increased but, as the H⁺‐ATPase is electrogenic, the increased ion permeability would allow a higher rate of H⁺ efflux. The AP‐3 adaptor complex is involved in traffic from Golgi to vacuoles but its function in plants is not much known. The phenotypes of the wat1‐1D mutant can be explained if loss of function of the AP‐3 β‐adaptin causes activation of channels or transporters for organic anions (acetate) and for K⁺ at the plasma membrane, perhaps through miss‐localization of tonoplast proteins. This suggests a role of this adaptin in trafficking of ion channels or transporters to the tonoplast.     

Separation of D-forms and L-forms of DL-aspartic acid derivatives by the enzyme subtilisin DY

The ability of the enzyme subtilisin DY for the synthesis of derivatives of DL-aspartic acid which are differently N and C-terminal protected and semiproducts of the peptide synthesis was investigated. The enzyme reaction was characterized by high yields and a comparatively short reaction time. Two of the substrates, Z-D,L-Asp-(OMe)₂ and PhAc-D,L-Asp-(OMe)₂, were hydrolyzed for about 15 min; the reaction time for Boc-D,L-Asp-(OMe)₂ was 2.5 h. The values for the MICHAELIS constants obtained for Z-D,L-Asp-(OMe)₂ (K_m = 0.576 mM) and PhAc-D,L-Asp-(OMe)₂ (K_m = 0.300 mM) showed a high affinity of the enzyme to the substrates. For Boc-D,L-Asp-(OMe)₂ the affinity of the enzyme is considerable lower (K_m = 14.07 mM). The results of these investigations can be effectively used for the separation of N-protected derivatives of D,L-aspartic acid and with a high probability also for other amino and racemic forms.     

Isolation and Characterization of a Cellobiose Dehydrogenase Formed by a Nonsporulating Mycelial Fungus INBI 2-26(-)

A nonsporulating fungus isolated from dioxin-containing tropical soils forms cellobiose dehydrogenase when grown in media supplemented by a source of cellulose. The enzyme purified to homogeneity by SDS-PAGE (yield, 43%) had an M_r of 95 kDa; its pH optimum was in the range 5.5–7.0; more than 50% activity was retained at pH 4.0–8.0 (citrate–phosphate buffer). The absorption spectrum of the enzyme in the visible range had the characteristic appearance of flavocytochrome proteins. Cellobiose dehydrogenase oxidized cellobiose and lactose (the respective K _M values at pH 6.0 equaled 4.5 ± 1.5 and 56 M) in the presence of dichlorophenolindophenol (K _M,_app = 15 ± 3 M at pH 6.0) taken as an electron acceptor. Other sugars were barely if at all oxidized by the enzyme. Neither ethyl--D-cellobioside, heptobiose, nor chitotriose inhibited the enzymatic oxidation of lactose, even under the conditions of 100-fold molar excess. The enzyme was weakly inhibited by sodium azide dichlorophenolindophenol reduction and exhibited an affinity for amorphous cellulose. At 55°C and pH 6.0 (optimum stability), time to half-maximum inactivation equaled 99 min. The enzyme reduced by cellobiose was more stable than the nonreduced form. Conversely, the presence of an oxidizer (dichlorophenolindophenol) decreased the stability eight times at pH 6.0. In addition, the enzyme acted as a potent reducer of the one-electron acceptor cytochrome c ³⁺ (K _{M app} = 15 M at pH 6.0).     

Diffusion kurtosis imaging in head and neck cancer: A correlation study with dynamic contrast enhanced MRI

PurposeTo investigate the biophysical meaning of Diffusion Kurtosis Imaging (DKI) parameters via correlations with the perfusion parameters obtained from a long Dynamic Contrast Enhanced MRI scan, in head and neck (HN) cancer.MethodsTwenty two patients with newly diagnosed HN tumor were included in the present retrospective study. Some patients had multiple lesions, therefore a total of 26 lesions were analyzed. DKI was acquired using 5b values at 0, 500, 1000,1500 and 2000 s/mm². DCE-MRI was obtained with 130 dynamic volumes, with a temporal resolution of 5 s, to achieve a long scan time (>10 min). The apparent diffusion coefficient D_app and apparent diffusional kurtosis K_app were calculated voxel-by-voxel, removing the point at b value = 0 to eliminate possible perfusion effects on the parameter estimations. The transfer constants K^trans and K_ep, v_e, and the histogram-based entropy (En) and interquartile range (IQR) of each DCE-MRI parameter were quantified. Correlations between all variables were investigated by the Spearman’s Rho correlation test.ResultsModerate relationships emerged between D_app and K_ep (Rho =  − 0.510, p = 0.009), and between D_app and v_e (Rho = 0.418, p = 0.038). En(K_ep) was significantly related to K_app (Rho = 0.407, p = 0.043), while IQR(K_ep) showed an inverse association with D_app (Rho = -0.422, p = 0.035).ConclusionsA weak to intermediate correlation was found between DKI parameters and both K_ep and v_e. The kurtosis was associated to the intratumoral heterogeneity and complexity of the capillary permeability, expressed by En(K_ep).     

Diffusion properties of bacteriophages through agarose gel membrane

A simple two‐chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D_app) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to be much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G, and tetracycline. D_app of T4 and fNEL in water through filter paper were calculated to be 2.8 × 10^?11 m²/s and 6.8 × 10^?12 m²/s, respectively, and D_app of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D_app of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D_app of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Influences of nonuniform activity distributions on the apparent maximum reaction rate and apparent Michaelis constant of immobilized enzyme reactions

The influences of nonuniform activity distribution within a porous solid support on the apparent kinetic parameters, V_m^app and K_m^app, of immobilized enzyme reactions following the Michaelis-Menten kinetics were theoretically investigated. As the enzyme is distributed to the neighborhood of the external surface of the support, V_m^app and K_m^app approach their respective intrinsic values over a wide range of substrate concentration. There is a close relationship between the nonuniform distribution and internal diffusional resistance. Changes in these two factors provide similar effects on V_m^app and K_m^app. As long as the immobilized enzyme reaction follows Michaelis-Menten kinetics, the nonuniform activity distribution never makes K_m^app less than its intrinsic value.