Showed Saturation Kinetics Research Articles

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g−1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8–9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a Km of 6.6 mM for H2O2 and Vmax of 707 mM H2O2 min−1 g−1 wet wt. cells, and showed saturation kinetics at 50 mM H2O2. The cells were entrapped in calcium alginate and used for H2O2 degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.

Characteristics of (+)-Catechin and (–)-Epicatechin Transport across Pig Intestinal Brush Border Membranes

Background/Aims: (+)-Catechin and (–)-epicatechin are considered as disease preventive flavan-3-ols of foods like fruits, beverages and chocolate. We investigated mechanisms and kinetics of (+)-catechin and (–)-epicatechin uptake employing a validated in vitro model with isolated pig brush border membrane vesicles. Methods: Vesicles were isolated from pig small intestine employing the divalent cation method. Characterization (marker enzymes, electron microscopy) confirmed their purity and function. Transport studies with (+)-catechin and (–)-epicatechin under predefined conditions [presence/absence of sodium, pH gradient, temperature (8–37°C), various initial substrate concentrations (2–20 mmol/l)] revealed a measurable transport (HPLC analyses) across the brush border membrane for both substrates. Re sults: Catechin transport was stimulated by an outwardly directed H⁺ gradient (pH_i 5.5/pH_o 7.5). The presence of an inwardly directed Na⁺ gradient did not result in a transient overshoot in (+)-catechin and (–)-epicatechin uptake. At 37°C, subtraction of diffusion from the total transport rate showed saturation kinetics. Conclusion: Our in vitro study indicate that both (+)-catechin and (–)-epicatechin are transported across the basolateral membrane using a dual transport system consisting of free diffusion (dominant at low concentrations) and carrier-mediated facilitated diffusion.

Mitochondrial transport in proline catabolism in plants: the existence of two separate translocators in mitochondria isolated from durum wheat seedlings

Abiotic stresses, such as high salinity or drought, can cause proline accumulation in plants. Such an accumulation involves proline transport into mitochondria where proline catabolism occurs. By using durum wheat seedlings as a plant model system, we investigated how proline enters isolated coupled mitochondria. The occurrence of two separate translocators for proline, namely a carrier solely for proline and a proline/glutamate antiporter, is shown in a functional study in which we found the following: (1) Mitochondria undergo passive swelling in isotonic proline solutions in a stereospecific manner. (2) Externally added L: -proline (Pro) generates a mitochondrial membrane potential (Delta Psi) with a rate depending on the transport of Pro across the mitochondrial inner membrane. (3) The dependence of the rate of generation of Delta Psi on increasing Pro concentrations exhibits hyperbolic kinetics. Proline transport is inhibited in a competitive manner by the non-penetrant thiol reagent mersalyl, but it is insensitive to the penetrant thiol reagent N-ethylmaleimide (NEM). (4) No accumulation of proline occurs inside the mitochondria as a result of the addition of proline externally, whereas the content of glutamate increases both in mitochondria and in the extramitochondrial phase. (5) Glutamate efflux from mitochondria occurs at a rate which depends on the mitochondrial transport, and it is inhibited in a non-competitive manner by NEM. The dependence of the rate of glutamate efflux on increasing proline concentration shows saturation kinetics. The physiological role of carrier-mediated transport in the regulation of proline catabolism, as well as the possible occurrence of a proline/glutamate shuttle in durum wheat seedlings mitochondria, are discussed.

Synthesis, characterisation and catalase-like activity of dimanganese(III) complexes of 1,5-bis(5-X-salicylidenamino)pentan-3-ol (X = nitro and chloro)

The dimanganese(III,III) complexes [ Mn 2 III ( 5 - NO 2 - salpentO ) ( μ - AcO ) ( μ - MeO ) ( methanol ) 2 ] Y ( 1: Y = Br, 2a: Y = I, 2b: Y = I 3), [ Mn 2 III ( 5 - NO 2 - salpentO ) ( μ - AcO ) ( μ - MeO ) ( methanol ) ( ClO 4 ) ] ( 3) and [ Mn 2 III ( 5 - Cl - salpentO ) ( μ - AcO ) ( μ - MeO ) ( methanol ) 2 ] Br ( 4), where salpentOH is the symmetrical Schiff base ligand 1,5-bis(salicylidenamino)pentan-3-ol, were synthesised and structurally characterized. Complex 2b crystallises in the monoclinic system, space group P2 1/c, and exhibits Mn⋯Mn separation of 2.911 Å. This Mn⋯Mn separation is very close to the other characterized ( μ - alkoxo ) 2 ( μ - acetato ) Mn 2 III complexes of X-salpentOH (X = MeO, Br and H) and reveals that the aromatic substituent has little influence on the geometric parameters of the bimetallic core. A correlation between the electronic character of the different ring substituents, the redox potentials of the dinuclear complexes and their catalase activity was evidenced. Complexes 1– 4 show saturation kinetics with [H 2O 2] and the H 2O 2 disproportionation involves redox cycling between the Mn 2 III / Mn 2 IV levels. The catalytic activity studies show that bound acetate is required for catalase activity and that the acetato and alkoxo bridges serve as internal bases facilitating the proton transfer coupled to oxidation of the metal centre.

Enantioselective epoxidation of chalcone catalysed by the artificial enzyme poly- l-leucine: kinetic mechanism

An insight into the kinetics, mechanism and optimum reaction conditions of the Juliá-Colonna enantioselective epoxidation has been gained using a soluble polyleucine catalyst (PLL), chalcone as the substrate, and hydrogen peroxide (HOO −) as the oxidant. PLL shows saturation kinetics for both chalcone and HOO − and has a behaviour that fits a steady state random bireactant system with one of the pathways (HOO − binding first) being kinetically preferred to the other (chalcone binding first) in the formation of the ternary complex PLL:HOO −:Chalcone.

Increasing magnesium intakes in relation to magnesium absorption in dry cows.

Earlier studies with temporarily isolated rumen of heifers show saturation kinetics of Mg efflux across the rumen wall. Therefore, we hypothesized that high Mg intakes would not further increase the rate of Mg absorption in cows. To test our hypothesis, six ruminally fistulated non-pregnant dry cows were given diets with different Mg concentrations in a 6 x 6 Latin square design. Desired concentrations of Mg were attained by adding MgO to the basal diet and the Mg concentrations in the total rations were 3.8, 6.4, 9.1, 11.8, 14.1 and 173 g Mg/kg dry matter, which provided Mg intakes of 27.1, 44.6, 64.6, 83.5, 100.4 and 124.3 g/d, respectively. Increasing Mg intakes were associated with increased (P<0.001) faecal Mg excretion. However, apparent Mg absorption expressed as g/d was not significantly different for Mg intakes from 100.4 to 124.3 g/d while Mg absorption expressed as a proportion of intake was not significantly different for Mg intakes ranging from 64.6 to 124.3 g/d. Mg concentrations in rumen fluid after feeding increased (P<0.001) with increasing Mg intakes. Apparent absorption of Mg appeared to become saturated at a ruminal Mg concentration of 17.5 mM (Mg intake of 83.5 g/d). Group-mean post-feeding concentrations of Mg and Na in rumen fluid were significantly correlated (Pearson's r=-0.96; P=0.003, n=6). This study showed that under conditions of practical dairy cow feeding, Mg absorption was maximal at Mg intakes > or = 84 g/d.

Glyoxalase II of African Trypanosomes Is Trypanothione-dependent

The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.

Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence

The fluorogenic properties of quercetin and similar flavonoids common in plants were exploited to analyse their interaction with target proteins. Quercetin produced a strong fluorescent signal upon binding to bovine serum albumin (BSA) and insulin. The fluorescent signal showed saturation kinetics with increasing flavonoid concentrations indicating the presence of defined peptide binding motifs. Other tested proteins showed no fluorescence with the flavonoids. In a comparative study including 22 flavonoids the compounds with fluorogenic properties were identified using our model proteins BSA and insulin and the structural requirements for the fluorogenic property were defined. Only flavones with a high degree of hydroxylation were able to elicit fluorescence. The emitted fluorescence was strongly enhanced at alkaline pH. Finally, an attempt was made to identify intracellular target molecules in live cells. Drosophila follicles showed a distinct staining pattern thus giving evidence that high concentrations of quercetin binding proteins are present in the nuclei and are associated with the ring canals. The presented biochemical and cytological data show that the interaction of the studied flavonoids with target proteins is specific and this finding opens up new experimental possibilities to systematically identify the cellular proteins with specific binding motifs for quercetin or other fluorogenic compounds of medical interest.

Cadmium uptake in rat hepatocytes in relation to speciation and to complexation with metallothionein and albumin.

Cadmium (Cd) uptake has been studied in primary cultures of rat hepatocytes focusing on the impact of inorganic and organic speciation. Uptake time-course studies over a 60-min exposure to 0.3 microM (109)Cd revealed a zero-time uptake and a slower process of accumulation which proceeds within minutes. (109)Cd uptake showed saturation kinetics (K(m) = 3.5 +/- 0.8 microM), and was highly sensitive to inhibition by Zn and Hg. There was no evidence for sensitivity to the external pH nor for any preferential transport of the free cation Cd(2+) over CdCl(n) (2-n) chloro-complexes. According to the assumption that only inorganic metal species are available, metal uptake decreased upon albumin (BSA) addition to the exposure media. In contrast, higher levels of (109)Cd accumulation were obtained under optimal conditions for Cd complexation by MT. Comparison among uptake data obtained under inorganic and organic conditions revealed that Cd-MT would be taken up 0.4 times as rapidly as Cd(inorg). We conclude that uptake of Cd in rat hepatocytes involves specific transport mechanism(s) subjected to Zn or Hg interactions. Uptake of inorganic Cd is not proportional to the levels of free Cd(2+) and does not involve the divalent cation transporter DCT1 nor the co-transporter Fe(2+)-H(+) NRAMP2. We found Cd-MT but not Cd-BSA to be available for the liver cells, and have estimated a binding affinity four orders of magnitude higher for Cd complexation with MT compared to BSA; MT may have a significant role in Cd delivery to the liver.

녹조 납작파래 (Enteromorpha compressa)의 질산염 흡수, 배아 생장 및 지방산 조성에 대한 광 및 온도의 영향

Effects of light and temperature on the nitrate uptake and germling growth of Enteromorpha compressa (L.) Greville (Chlorophyta) were studied based on samples from Cheongsapo near Busan, Korea. In addition, their effects on fatty acids composition in thallus were examined. Nitrate uptake showed saturation kinetics. <TEX>$V_{max}$</TEX> (maximal uptake rate) and its <TEX>$K_s$</TEX> (half-saturation constant) at <TEX>$20^{\circ}C,\;80\;{\mu}mol\;m^{-2}s^{-1},$</TEX> white light were <TEX>$1.571\;{\mu}mol{\cdot}g\;fr\;wt^{-1}{\cdot}h^{-1}$</TEX> and 3.56 <TEX>${\mu}M$</TEX>, respectively. In nitrate uptake with irradiance, wavelength and temperature, its rate represented respectively the highest value as <TEX>$1.405\pm0.020,\;0.623\pm0.040,\;1.422\pm0.022\;{\mu}mol{\cdot}g\;fr\;wt^{-1}{\cdot}h^{-1}\;at\;100\;{\mu}mol\;m^{-2}s^{-1},$</TEX> red light, <TEX>$20^{\circ}C$</TEX> and exhibited significant difference among the examined conditions (p<0.001). Germling growth of E. compressa also showed saturation kinetics, and <TEX>$V_{max}$</TEX> and its <TEX>$K_s$</TEX> value at <TEX>$20^{\circ}C,\;100\;{\mu}mol\;m^{-2}s^{-1},$</TEX> 12:12 h were <TEX>$56.18\%\;day^{-1}$</TEX> and 0.33 <TEX>${\mu}M$</TEX>, respectively. SGR (specific growth rate) recorded a maximal value as 49.33-54.80, 39.07-50.72, <TEX>$47.20-54.53\%\;dat^{-1}$</TEX> at <TEX>$120\;{\mu}mol\;m^{-2}s^{-1},$</TEX> blue light and <TEX>$18^{\circ}C$</TEX> respectively, and showed significant difference (p<0.001). Red light made the effective nitrate uptake, but germling growth was largely limited by the light. In fatty acids analysis, PUFAs (polyunsaturated fatty acids) were high at blue light, <TEX>$18^{\circ}C,\;100\;{\mu}M\;NO_3^-.$</TEX> However, irradiance did not affect the production of PUFAs. In conclusion, nitrate uptake and germling growth of E. compressa showed saturation kinetics to external nitrate concentration, and were significantly affected by irradiance, wave length and temperature. Fatty acid composition was also influenced by the factors except for irradiance. Their maximal values, together with the highest production of PUFAs, were found at blue light band, <TEX>$20^{\circ}C,\;100\;{\mu}mol\;m^{-2}s^{-1},\;and\;100\;{\mu}M\;NO_3^-.$</TEX>

Tuning the Catalase Activity of Dinuclear Manganese Complexes by Utilizing Different Substituted Tripodal Ligands

AbstractA series of five new dinuclear manganese(II/II) compounds with derivatives of 2‐{[bis(pyridin‐2‐ylmethyl)amino]methyl}phenol (HL1) were synthesized and structurally characterized. All complexes crystallize in monoclinic space groups and exhibit Mn···Mn separations in the range of 3.392(8)−3.493(2) Å caused by bis(µ‐phenoxo) bridging modes. The derivatives of HL1 contain either electron‐donating or ‐withdrawing substituents. A correlation between the electronic character of the different ring substituents, the redox potentials of the dinuclear transition metal complexes and the catalase activity has been revealed. In analogy to manganese catalase, all complexes show saturation kinetics at high substrate concentration. The investigated catalase activity places the compounds in the upper range of functional catalase mimics. (© Wiley‐VCH Verlag GmbH &amp; Co. KGaA, 69451 Weinheim, Germany, 2004)

Saturable, energy-dependent uptake of phenanthrene in aqueous phase by Mycobacterium sp. strain RJGII-135.

The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-(14)C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state (14)C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K(t)) of 26 +/- 3 nM (mean +/- standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated (14)C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K(d)) of 41 +/- 21 nM (mean +/- standard deviation). Given the low values of K(t) and K(d), Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.

D-Glucose uptake in fish hepatocytes: mediated by transporter in rainbow trout ( Oncorhynchus mykiss), but only by diffusion in prespawning lamprey ( Lampetra fluviatilis) and in RTH-149 cell line

The transport of d-glucose into rainbow trout ( Oncorhynchus mykiss) and river lamprey ( Lampetra fluviatilis) hepatocytes, as well as into rainbow trout hepatoblastoma cell line RTH-149 was studied using tracer methods. The half-time for d-glucose equilibration was 15 s for rainbow trout. The half-times for the non-metabolizable d-glucose analog, 3- O-methyl- d-glucose equilibration were 8 s, 37 s and 38 s for rainbow trout, lamprey and RTH-149 cells, respectively. The 3- O-methyl- d-glucose was taken up by rainbow trout hepatocytes by facilitated diffusion in addition to simple diffusion. The uptake showed saturation kinetics with the K m of 37 mM and V max of 62 mmol kg −1 cells min −1. The uptake was sensitive to phloretin and cytochalasin B, but not affected by ouabain. The 3- O-methyl- d-glucose uptake by lamprey hepatocytes and RTH-149 cells showed no indication of saturation up to 160 mM, and was not affected by phloretin, cytochalasin B or ouabain, which suggests the mode of transport to be by passive diffusion. However, immunocytochemical stainings revealed the existence of mammalian type GLUT1 and GLUT2 transporters in all cells studied. The lack of a functioning carrier-mediated glucose uptake in lamprey hepatocytes might be due to its physiological state (prespawning starvation). The minor 3- O-methyl- d-glucose uptake into RTH-149 cells compared to freshly isolated rainbow trout hepatocytes might reflect low metabolic activity of the cell lines. Under the conditions applied the RTH-149 cell line is no suitable in vitro model for glucose transport in fish cells.

Vacuolar malate uptake is mediated by an anion-selective inward rectifier.

Electrophysiological studies using the patch-clamp technique were performed on isolated vacuoles from leaf mesophyll cells of the crassulacean acid metabolism (CAM) plant Kalanchoë daigremontiana to characterize the malate transport system responsible for nocturnal malic acid accumulation. In the presence of malate on both sides of the membrane, the current-voltage relations of the tonoplast were dominated by a strongly inward-rectifying anion-selective channel that was active at cytoplasmic-side negative voltages. Rectification of the macroscopic conductance was reflected in the voltage-dependent gating of a 3-pS malate-selective ion channel, which showed a half-maximal open probability at -43 mV. Also, the time-averaged unitary currents following a step to a negative voltage corresponded to the time-dependent kinetics of the macroscopic currents, suggesting that the activity of this channel underlies the anion-selective inward rectifier. The inward rectifier showed saturation kinetics with respect to malate (apparent Km of 2.5 mm malate2- activity), a selectivity sequence of fumarate2- > malate2- > Cl- > maleate2- approximately citrate3-, and greater activity at higher pH values (with an apparent pK of 7.1 and maximum activity at around pH 8.0). All these properties were in close agreement with the characteristics of malate transport observed in isolated tonoplast vesicles. Further, 100 microM niflumate reversibly blocked the activity of the 3-pS channel and inhibited both macroscopic currents and malate transport into tonoplast vesicles to the same extent. The macroscopic current densities recorded at physiological voltages and the estimated channel density of 0.2 microm-2 are sufficient to account for the observed rates of nocturnal malic acid accumulation in this CAM plant, suggesting that the 3-pS, inward-rectifying, anion-selective channel represents the principal pathway for malate influx into the vacuole.

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.

Spectrophotometric investigations with hexa-coordinate ferric lignin peroxidase: does water retention at the active site influence catalysis?

Native lignin peroxidase (LIP) can adopt either a stable penta- or hexa-coordinate state. We have examined catalysis with hexa-coordinate ferric LIP as the starting material, using rapid scanning spectrophotometry. Initial two-electron oxidation of hexa-coordinate native LIP by H 2O 2 (Compound I formation) was accompanied by a shifting isosbestic point (419 → 416 nm), consistent with displacement of a resident water molecule, prior to the reaction of the ferric iron with H 2O 2. The Compound I species derived from a hexa-coordinate ferric state shows an unusual peak at 520 nm, which may be due to water retention in the vicinity of the heme active site. Compound I reduction by veratryl alcohol showed saturation kinetics, which contrasts with the situation observed when Compound I is derived from a penta-coordinate ferric state. The data inferred that water can interfere with heme access by electron donors, altering the mechanism of Compound I reduction.

Complete characterisation of Tn 5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism

The complete genetic characterisation of Tn 5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn 3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn 5530 is terminated at both ends by the IS 1071::IS 1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn 5530 sequence length, including the IS 1071::IS 1471 elements, was found to be 40,956 bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1β subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N, N ′-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.

Characterization of cholesterol transport from midgut to fat body in Manduca sexta larvae

Using in vitro methods, we investigated the transfer of cholesterol from larval Manduca sexta midgut to the hemolymph lipoprotein, lipophorin, and the transfer of cholesterol from lipophorin to larval fat body. In the midgut, transfer of free cholesterol shows saturation kinetics, but the apparent Km is higher than the measured Kd for the midgut lipophorin-receptor complex. In addition, the transfer is unaffected by suramin, which binds to the receptor and inhibits lipophorin binding, and by antibodies to the lipid transfer particle, which is required for export of diacylglycerol from the midgut to lipophorin. In the fat body, transfer of free cholesterol also shows saturation kinetics, and the apparent Km is higher than the measured Kd for the fat body lipophorin–receptor complex. Suramin and anti-lipid transfer particle antibodies exert only a small (20%) inhibitory effect. In both tissues it seems that the most likely mode of cholesterol transfer is via aqueous diffusion, which is also an important mechanism in vertebrate cells. Based on these results, we propose that cholesterol homeostasis in larval M. sexta is maintained by a mass action mechanism in which cholesterol is freely transferred between lipophorin and tissues depending on the needs of the tissues. This simple mechanism is ideally suited to insects, which can neither make cholesterol nor internalize lipophorin, the two mechanisms that vertebrate cells use to control their cholesterol content.

MRI and fluorescence microscopy of the acute vascular response to VEGF165: vasodilation, hyper-permeability and lymphatic uptake, followed by rapid inactivation of the growth factor.

Vascular endothelial growth factor (VEGF) is one of the key growth factors regulating tumor angiogenesis and thus it is one of the primary targets for antiangiogenic therapy. The long-term effects of VEGF include induction of proliferation and migration of endothelial cells, tube formation and maintenance of the immature capillaries. The early effects of VEGF include vasodilation and increased permeability. We hypothesize that the early responses to VEGF can serve to develop a quantitative measure of the activity of VEGF, and therefore may be applicable for monitoring the efficacy of systemic suppression of VEGF signaling during antiangiogenic therapy. For that end we tested the ability of MRI and fluorescence microscopy to detect the early response to intradermal VEGF165 in nude mice. VEGF-induced local vasodilation and increased permeability was detected by intravenous administration of macromolecular biotin-BSA-GdDTPA(23) 30 min after intradermal administration of VEGF. Contrast leak showed saturation kinetics. Delayed contrast administration (90 min after intradermal administration of VEGF) resulted in low contrast leak and demonstrated that the saturation kinetics is not due to contrast equilibration between plasma and the interstitial space, but rather is due to suppression of vascular permeability. Permeability was restored by a second bolus of VEGF, showing that the saturation kinetics is primarily due to inactivation of the growth factor. Confocal microscopy of fluorescent BSA-FITC confirmed the permeability changes monitored by MRI. Moreover, confocal microscopy showed efficient lymphatic uptake of the extravasated contrast material specifically in regions of VEGF induced hyper-permeability.

Synthesis and characterisation of dimanganese(iii) complexes of 1,5-bis(X-salicylidenamino)pentan-3-ol (X = 3- or 5-methoxy) and their catalytic activity towards hydrogen peroxide disproportionation

Structural, magnetic, electrochemical, EPR, 1H NMR, and H2O2 reactivity studies are reported for [MnIII2(X-salpentO)(μ-AcO)(μ-MeO)(MeOH)2]Br (1: X = 5-OMe and 2: X = 3-OMe; salpentOH = 1,5-bis(salicylidenamino)pentan-3-ol). The X-ray diffraction analysis of 2 reveals that the manganese atoms possess an axially elongated-octahedral geometry. The elongation is perpendicular to the bridging plane resulting in a short Mn⋯Mn distance of 2.9278(6) A. The ligand lies in the meridional plane and the sixth co-ordination position of each manganese atom is occupied by a methanol molecule providing two substitution-labile sites in cis-position. Solution structural studies indicate that complexes 1 and 2 are stable in methanol and dmf, even in the presence of water, and the solid state structure is retained in solution. The two complexes show catalytic activity toward disproportionation of H2O2 in methanol and dmf at 25 °C. The initial rate of oxygen evolution in the presence of 1 or 2 is first order in [catalyst] and show saturation kinetics with [H2O2]. Complexes 1 and 2 dismutate H2O2 without a kinetic lag phase and are implicated as the active form of the reduced catalyst. Comparison to other dimanganese complexes of this family reveals that the methoxy phenyl-ring substituted derivatives are more effective at reducing H2O2 but are less efficient at binding the H2O2 substrate (higher KM) than the 5-bromo or the unsubstituted complexes.