The role of the neurotransmitters acetylcholine and noradrenaline in the pathogenesis of stress ulcers

Molecular self-diffusion coefficients of water (0.75 +/- 0.05), N-acetylaspartate (0.27 +/- 0.04), creatines (0.27 +/- 0.04), and cholines (0.28 +/- 0.08) x 10(-5) cm2 s-1 were obtained from localized proton NMR spectra of rat brain in vivo using diffusion-weighted stimulated-echo (STEAM) sequences with a diffusion time of (delta--delta/3) = 17 ms.     

Macromolecular permeability across the blood-nerve and blood-brain barriers

The permeability of insulin (Ins), nerve growth factor (NGF), albumin (Alb), transferrin (Trf), and IgG across the blood-nerve barrier (BNB) and blood-brain barrier (BBB) in normal adult rats was quantified by measuring the (permeability coefficient x surface area) product (PS) with the i.v. bolus-injection technique in the cannulated brachial vein and artery using radioiodinated proteins. The PS values of the BNB for IgG and Alb were low: 0.079 +/- 0.029 x 10(-6) and 0.101 +/- 0.088 x 10(-6) ml.g-1.s-1, (mean +/- SD, respectively). The PS values for NGF and Trf were 16.1-fold and 25.5-fold higher than for Alb. The PS for Ins across the BNB was 33.190 +/- 2.053 x 10(-6) ml.g-1.s-1--a remarkable 329-fold increase compared with Alb. The PS values of the BBB for IgG and Alb in different brain regions were all low, from 0.028 +/- 0.017 to 0.151 +/- 0.035 x 10(-6) ml.g-1.s-1 (mean +/- SD). NGF and Trf had comparable PS values from 13- to 32-fold higher than for Alb, except for the brain stem, where the PS for Trf was 66-fold higher than for Alb. The mean PS for Ins across the BBB ranged from 15.78 +/- 5.45 x 10(-6) ml.g-1.s-1 for the cortex to 22.62 +/- 7.50 x 10(-6) ml.g-1.s-1 for the brain stem--again a remarkable 105- to 390-fold increase relative to Alb. Because reliable PS measurements were obtained for all proteins tested, the BBB and BNB cannot be considered impermeable to proteins--a concept that has plagued brain- and nerve-barrier research. The low PS values for IgG and Alb indicate low rates of transfer; however, Alb, in particular, is the major protein of endoneurial and ventricular fluid, which suggests that these PS values may be significant. Ins had the highest PS values, which likely reflect the mechanism of transport across the barriers--that is, receptor-mediated transport. Because NGF and Trf had PS values 13- to 66-fold higher than for Alb, whether this reflects receptor-mediated uptake, adsorptive-mediated transcytosis, or some other mechanism is unclear. That the PS values for NGF and Trf differ from Alb and IgG clearly suggests, however, a different uptake mechanism. Finally, the remarkably high PS values for Ins across the BBB and BNB identify this protein and its putative receptor on capillary endothelial cells as a potential target for drug delivery into the central and peripheral nervous systems.     

Transferrins. Hen ovo-transferrin, interaction with bicarbonate and iron uptake

Fe(III) uptake by the iron-delivery and iron-scavenging protein, hen ovotransferrin has been investigated in vitro between pH 6.5 and 9. In the absence of any ferric chelate, apo-ovotransferrin loses two protons with K1a = 50 +/- 1 nM and K2a = 4.0 +/- 0.1 nM. These acid-base equilibria are independent of the interaction of the protein with bicarbonate. The interaction with bicarbonate occurs with two different affinity constants, KC = 9.95 +/- 0.15 mM and KN = 110 +/- 10 mM. FeNAc3 exchanges its Fe(III) with the C-site of the protein in interaction with bicarbonate, direct rate constants k1 = 650 +/- 25 M-1 s-1, reverse rate constant k-1 = (6.0 +/- 0.1) x 10(3) M-1 s-1 and equilibrium constant K1 = 0.11 +/- 0.01. This iron-protein intermediate loses then a single proton, K3a = 3.50 +/- 0.35 nM, and undergoes a first change in conformation followed by a two or three proton loss, first order rate constant k2 = 0.30 +/- 0.01 s-1. This induces a new modification in conformation followed by the loss of one or two protons, first order rate constant k3 = (1.50 +/- 0.05) x 10(-2) s-1. These modifications in the monoferric protein conformation are essential for iron uptake by the N-site of the protein. In the last step, the monoferric and diferric proteins attain their final state of equilibrium in about 15,000 s. The overall mechanism of iron uptake by ovotransferrin is similar but not identical to those of serum transferrin and lactoferrin. The rates involved are, however, closer to lactoferrin than serum transferrin, whereas the affinities for Fe(III) are lower than those of serum transferrin and lactoferrin. Does this imply that the metabolic function transferrins is more related to kinetics than to thermodynamics?     

Transport of quercetin and its glucosides across human intestinal epithelial Caco-2 cells

There is mounting evidence from human epidemiological, animal in vivo, and in vitro studies to suggest beneficial effects related to the consumption of quercetin and its glucosides. However, there is limited knowledge on the oral bioavailability of these natural products. This study examined the intestinal epithelial membrane transport of quercetin, quercetin 4'-glucoside, and quercetin 3,4'-diglucoside, using the Caco-2 human colonic cell line, a model of human intestinal absorption. The apparent permeability (Papp) of each agent was measured in both apical to basal and basal to apical directions. The apical to basolateral flux of quercetin, Papp 5.8 +/- 1.1 x 10(-6) cm x sec(-1) (mean +/- SEM), was more than 10-fold higher than for the paracellular transport marker mannitol, 0.48 +/- 0.09 x 10(-6) cm x sec(-1) (P < 0.01). Under identical conditions, the Papp for the transcellular marker propranolol was about 5-fold higher than for quercetin (P < 0.001). Interestingly, the reverse, basolateral to apical, flux of quercetin (Papp 11.1 +/- 1.2 x 10(-6) cm x sec(-1)) was almost 2-fold higher than the apical to basolateral flux (P < 0.001). In similar experiments, quercetin 4'-glucoside demonstrated no absorption, Papp < 0.02 x 10(-6) cm x sec(-1) in the apical to basal direction, but did demonstrate basal to apical flux, Papp 1.6 +/- 0.2 x 10(-6) cm x sec(-1). Quercetin 3,4'-diglucoside showed a low apical to basolateral transport (Papp 0.09 +/- 0.03 x 10(-6) cm x sec(-1)); its reverse, basolateral to apical, transport was, however, 4-fold higher (P < 0.05). In these cells, glucose was actively transported with an apical to basolateral Papp of 36.8 +/- 1.1 x 10(-6) cm x sec(-1). These observations suggest facile absorption of quercetin through the human intestinal epithelium, but contrary to a previous proposal, they do not support an active transport process for quercetin glucosides.     

Mechanism of intestinal absorption of ranitidine and ondansetron: transport across Caco-2 cell monolayers

We have investigated the transport of ranitidine and ondansetron across the Caco-2 cell monolayers. The apparent permeability co-efficients (Papp) were unchanged throughout the concentration range studied, indicating a passive diffusion pathway across intestinal mucosa. No metabolism was observed for ranitidine and ondansetron during the incubation with Caco-2 cell monolayers. Papp values for ranitidine and ondansetron (bioavailability of 50 and approximately 100% in humans, respectively) were 1.03 +/- 0.17 x 10(-7) and 1.83 +/- 0.055 x 10(-5) cm/sec, respectively. The Papp value for ranitidine was increased by 15- to 20-fold in a calcium-free medium or in the transport medium containing EDTA, whereas no significant change occurred with ondansetron, indicating that paracellular passive diffusion is not rate determining for ondansetron. Uptake of ondansetron by Caco-2 cell monolayers was 20- and 5-fold higher than that of ranitidine when the uptake study was carried out under sink conditions and at steady state. These results suggest that ranitidine and ondansetron are transported across Caco-2 cell monolayers predominantly via paracellular and transcellular pathways, respectively.     

Anisotropy of water diffusion in the myocardium of the rat

Pulsed field gradient nuclear magnetic resonance methods combined with nuclear magnetic resonance imaging were used to determine the water diffusion anisotropy in perfused rat hearts at 37 degrees C. It was found that the observed diffusion coefficient D(app) (apparent diffusion coefficient) depends on the orientation of the applied gradient g. When g is parallel to the epicardial surface, the observed diffusivity is D(app) parallel = 1.8 +/- 0.4 x 10(-9) m2.s-1, whereas when g is perpendicular to it, diffusivity is D(app) perpendicular = 2.5 +/- 0.5 x 10(-9) m2.s-1. To better characterize this directional dependence, images of the second-order diffusion tensor D of the myocardium were obtained. These data demonstrate several essential features of cardiac myoarchitecture, including the helicity of fiber orientation with respect to the ventricular axis and the variation of fiber pitch angle with transmural depth. Diffusion anisotropy may be quantified in a coordinate-independent manner by the eigenvalues of the diffusion tensor. In the myocardial midwall, these eigenvalues were E1 = 3.29 +/- 0.57, E2 = 2.01 +/- 0.42, and E3 = 0.77 +/- 0.58 x 10(-9) m2.s-1 (mean +/- SD). These data suggest that myocardial water diffusion is essentially unrestricted parallel to the myofibers. They further show that failure to measure the complete diffusion tensor may lead to substantial underestimates of diffusion anisotropy in the myocardium.     

Influence of apolipoprotein E polymorphism on the tracking of childhood levels of serum lipids and apolipoproteins over a 6-year period. The Bogalusa Heart Study

This study was designed to test the hypothesis that in the in vivo dog heart, increases in cyclic (c) GMP and also decreases in cAMP induced by intracoronary administration of acetylcholine are associated with depressed myocardial function. In 10 open-chest anesthetized dogs, 0.5 microgram.kg-1.min-1 of acetylcholine was infused into the left anterior descending coronary artery. The intracoronary infusion of acetylcholine was continued simultaneously with 0.1 microgram.kg-1.min-1 of isoproterenol. Regional segment work was calculated as the integrated product of force (auxotonic force transducer) and segment shortening (sonomicrometry). Regional myocardial O2 consumption was calculated from blood flow measurements and regional O2 saturations. Competitive radioligand binding assays were used to determine the intracellular level of cAMP and cGMP in the myocardium. Local intracoronary infusion of acetylcholine significantly reduced regional segment work (from 36.7 +/- 6.5 to 19.1 +/- 3.7 x 10(-3) J/min) and O2 consumption (from 6.4 +/- 0.8 to 3.8 +/- 0.7 mL O2.min-1.100 g-1). This was related to a decrease in cAMP levels (from 364 +/- 25 to 262 +/- 17 pmol/100 g) and an increase in cGMP levels (from 1.34 +/- 0.06 to 1.78 +/- 0.15 pmol/100 g). When isoproterenol (0.1 microgram.kg-1.min-1) was added to the acetylcholine infusion line, cAMP levels tripled to 769 +/- 84 pmol/100 g, while O2 consumption rose to 6.6 +/- 1.4 mL O2.min-1.100 g-1. However, regional work was only partially restored (25.7 +/- 4.8 x 10(-3) J/min). Thus, both cAMP decrements and cGMP elevation occurred together with the negative inotropic effect of acetylcholine, and increased cAMP alone (produced by isoproterenol) did not fully overcome the acetylcholine effect. This was associated with elevated intracellular levels of cGMP.     

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.     

Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique, and pulse radiolysis

Flash photolysis of alkaline peroxynitrite solutions results in the formation of nitrogen monoxide and superoxide. From the rate of recombination it is concluded that the rate constant of the reaction of nitrogen monoxide with superoxide is (1.9 +/- 0.2) x 10(10) M-1 s-1. The pKa of hydrogen oxoperoxonitrate is dependent on the medium. With the stopped-flow technique a value of 6.5 is found at millimolar phosphate concentrations, while at 0.5 M phosphate the value is 7.5. The kinetics of decay do not follow first-order kinetics when the pH is larger than the pKa, combined with a total peroxynitrite and peroxynitrous acid concentration that exceeds 0.1 mM. An adduct between ONOO- and ONOOH is formed with a stability constant of (1.0 +/- 0.1) x 10(4) M. The kinetics of the decay of hydrogen oxoperoxonitrate are not very pressure-dependent: from stopped-flow experiments up to 152 MPa, an activation volume of 1.7 +/- 1.0 cm3 mol-1 was calculated. This small value is not compatible with homolysis of the O-O bond to yield free nitrogen dioxide and the hydroxyl radical. Pulse radiolysis of alkaline peroxynitrite solutions indicates that the hydroxyl radical reacts with ONOO- to form [(HO)ONOO].- with a rate constant of 5.8 x 10(9) M-1 s-1. This radical absorbs with a maximum at 420 nm (epsilon = 1.8 x 10(3) M-1 cm-1) and decays by second-order kinetics, k = 3.4 x 10(6) M-1 s-1. Improvements to the biomimetic synthesis of peroxynitrite with solid potassium superoxide and gaseous nitrogen monoxide result in higher peroxynitrite to nitrite yields than in most other syntheses.     

Assessment of the degree of disability in narcolepsy

The effect of free fatty acids (FFA) and non-enzymatic glycation on the binding kinetics of dansylsarcosine (DS) to human serum albumin (HSA) was studied using the stopped-flow technique. The influence of FFA on the binding parameters of 25% glycated HSA depended on the type of fatty acid. The addition of stearic, oleic and linoleic acids in a concentration of 0.3 mmol/l showed no inhibitory effects on the association rate constant (k2) value for DS binding to 25% glycated HSA (k2 without FFA: 385 +/- 10 s-1, k2 with FFA > or = 385 +/- 10 s-1). In contrast, shorter chain fatty acids (hexanoic, octanoic, decanoic, lauric and myristic acids) showed marked inhibitory effects for 0.3 mmol/l FFA (k2 range: 233 +/- 32 to 69 +/- 5 s-1) and for 0.6 mmol/l FFA (k2 range: 125 +/- 3 to 20 +/- 4 s-1). The association rate constant (k2) as well as the affinity constant (KA) of DS were markedly affected by glycation: k2 was 686 +/- 61 s-1 for 7% glycated HSA, 385 +/- 10 s-1 for 25% glycated HSA and 209 +/- 12 s-1 for 50% glycated HSA. KA decreased from 6.1 +/- 2.9 x 10(5) M-1 for 7% glycated HSA, to 5.1 +/- 0.1 x 10(5) M-1 for 25% glycated HSA and to 1.3 +/- 0.6 x 10(5) M-1 for 50% glycated HSA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water transport in perfused scorpion ileum

Water transport in desert scorpion ileum involves two independent transfer pathways operating in parallel: 1) paracellular flow occurs through intercellular spaces in response to transmural osmotic or ionic gradients; and 2) transcellular water transport occurs across apical and basal cell membranes in response to a basal, energy-requiring sodium efflux process. The tissue exhibits no osmotic rectification over the range of transepithelial osmotic gradients imposed (Lp = hydraulic conductivity), Lp = 95 x 10(-7) cm - s-1 - atm-1), but displays apparent asymmetric ion permeability in response to transmural ion gradients, as determined by codiffusional water movements across the preparation. Osmotic permeability ((Pos), Pos = 1.13 x 10(-3) cm - s-1) of the tissue exceeds diffusional permeability ((Pd), Pd = 1.45 x 10(-5) cm - s-1) by almost two orders of magnitude. In the absence of osmotic or hydrostatic pressure gradients, transmural water transport requires cellular metabolism, is sodium-dependent, is inhibited by potassium, and produces an apparent strongly hypotonic absorbate. This water transport process appears to be adaptive, as scorpion dehydration results in alterations of luminal ion concentrations that favor increased net flow of water to the hemolymph.     

Preparation and characterization of biotinylated analogs of the calcium channel blocker omega-conotoxin

We have prepared a series of biotinylated analogs of omega-conotoxin (omega CgTx) as potent, selective markers for N-type calcium channels. At pH 9.5, reaction of omega CgTx with amidocaproylbiotin succinimidyl ester gives three biotinylated conjugates, labeled at lysines 2 or 24, or at both positions. Kinetic competition assays of 125I-omega CgTx binding to rat brain synaptic membranes show that each conjugate has a similar rate constant for association (1-1.3 x 10(6) M-1 s-1) but not dissociation (1-4 x 10(-4) s-1). Comparison with rate constants obtained for the association (1.2 x 10(7) M-1 s-1) and dissociation (5 x 10(-5) s-1) of native omega CgTx indicates that while biotinylation reduces omega CgTx potency (Kdkin = k-2/k2 = 4 pM for omega CgTx), binding of these labels to membranes is nevertheless of very high affinity (Kdkin 0.1-0.3 nM).     

The free solution mobility of DNA

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1 at 25 degrees C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from approximately 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes.     

Metmyoglobin/fluoride: effect of distal histidine protonation on the association and dissociation rate constants

The kinetics of formation and dissociation of the horse metmyoglobin/fluoride complex has been investigated between pH 3.4 and 11. The ionic strength dependence of the reaction has been measured at integral pH values between pH 5 and 10. Hydrofluoric acid, HF, binds to metmyoglobin with a rate constant of (4.7 +/- 0. 7) x 10(4) M-1 s-1. An apparent ionization in metmyoglobin with a pKa of 4.4 +/- 0.5 influences the rate of HF binding and is attributed to the distal histidine, His-64. Protonation of His-64 increases the HF binding rate by a factor of 2.6. The fluoride anion, F-, binds to metmyoglobin with a rate constant of (5.6 +/- 1.4) x 10(-2) M-1 s-1, about 10(6) times slower than HF. Binding of either HF or F- to hydroxymetmyoglobin cannot be detected. Protonation of the distal histidine facilitates HF dissociation from the metmyoglobin/fluoride complex. HF dissociates with a rate constant of 1.9 +/- 0.3 s-1. The fluoride anion dissociates 2000 times more slowly, with a rate constant of (8.7 +/- 1.6) x 10(-4) s-1. The apparent pKa for His-64 ionization in the fluorometmyoglobin complex is 5.7 +/- 0.1. The association and dissociation rate constants are relatively independent of ionic strength with secondary kinetic salt effects sufficient to account for the ionic strength variation of both, consistent with the idea that association and dissociation of neutral HF dominate the kinetics of fluoride binding to metmyoglobin.     

Influence of additives on the water uptake of hydrosilanized silicone rubbers

Water uptake characteristics of a silicone polymer containing additives of varying solubility have been investigated. A hydrophobic silica, a sparingly soluble agent and a soluble catalyst were added to a stoichiometrically balanced hydrosilanized silicone polymer (the inherent absorption of which was 0.09 wt%). The diffusion coefficients for desorption of all materials were of the order 10(-6) cm2s-1, whereas the diffusion coefficients for absorption showed a decrease with increasing hydrophilicity and solubility of the additive. The greater the solubility of the additive, the more prolonged the uptake and the greater the deviation from classic diffusion theory.     

The entry of [D-penicillamine2,5]enkephalin into the central nervous system: saturation kinetics and specificity

The delta opioid receptor-selective, enzymatically stable peptide [D-Penicillamine2,5]enkephalin (DPDPE) has recently acquired special significance with the identification of a saturable uptake system for this analgesic into the CNS. The aim of the present study was to characterize further the entry of [3H]DPDPE into the brain and CSF by means of a bilateral in situ brain perfusion method. Initial experiments revealed a saturable [3H]DPDPE uptake into the brain that followed Michaelis-Menten type kinetics with a K(m) value of 45.5 +/- 27.6 microM, a V(max) value of 51.1 +/- 13.2 pmol x min(-1) x g(-1) and a K(d) value of 0.6 +/- 0.3 microl x min(-1) x g(-1). Uptake of [3H]DPDPE into the CSF could not be inhibited (K(d) = 0.9 +/- 0.1 microl x min(-1) x g(-1)). Entry of [3H]DPDPE into the CNS was not inhibited in the presence of 10 mM 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH) or 50 microM ICI 174,864, which suggests that the saturable mechanism does not involve the large neutral amino acid transporter or binding to opioid receptors. It would also appear that [3H]DPDPE is not in competition with either poly-L-lysine or insulin to enter the CNS. However, both of these substances significantly increased the CNS entry of [3H]DPDPE but not that of the vascular space marker [14C]sucrose, and this may have valuable clinical implications. It is not known at present which saturable uptake mechanism is responsible for the CNS entry of [3H]DPDPE, but overall the results suggest a carrier-mediated transport system.     

Physiological transport properties of cultured retinal microvascular endothelial cell monolayers

PURPOSE: To characterize baseline transport properties: hydraulic conductivity (Lp), albumin permeability (Pe), and transendothelial electrical resistance (TER) of bovine retinal microvascular endothelial cells (RMEC) in the development of an in vitro model of the blood-retinal barrier (BRB). METHODS: RMEC were grown on porous, polycarbonate filters for determination of the number of days required to achieve minimal transport rates. Lp, Pe, and TER were measured by utilizing a bubble tracking spectrophotometer, by quantifying the diffusional movement of fluorescein isothiocyanate-labeled albumin, and by utilizing a Millipore electrical resistance meter, respectively. RESULTS: Lp decreased significantly from 7.82 +/- 0.85 x 10(-7) (mean +/- SEM) cm/sec/cm H2O at post-plating Day 5 to 1.44 +/- 0.26 x 10(-7) cm/sec/cm H2O at Day 9. Pe of the monolayer also decreased progressively with days post-plating from 3.44 +/- 0.53 x 10(-6) cm/sec at Day 7 to a minimum of 1.95 +/- 0.29 x 10(-6) cm/sec at Day II. Peak TER fluctuated until Day 7, when it began to steadily increase from 17.14 ohm-cm2 to a peak value of 25.42 ohm-cm2 at Day 10, decreasing from then on to 22.24 ohm.cm2 on Day 12. Known disrupters of the BRB, NECA and VEGF, elicited significant increase in RMEC Lp showing the sensitivity of this model to pharmacological alterations. CONCLUSIONS: Our data indicate that RMEC grown on polycarbonate filters form a restrictive monolayer of cells, which exhibit dynamic alterations in response to pharmacological agents, thus demonstrating an in vitro model of the BRB. Future studies with the model may offer insights into the pathogenesis of retinal vascular diseases and allow convenient testing of pharmacological interventions.     

Quantitation of beta 1 triiodothyronine receptor mRNA in human tissues by competitive reverse transcription polymerase chain reaction

Kinetics of photomodification of 26-meric deoxyribonucleotide pTTGCCTTGAATGGGAA-GAGGGTCATT with derivatives of the complementary oligonucleotides pTCTTCCCATTC, pTCTTCCCA, and pTTCCCA bearing a residue of (p-azidotetrafluorobenzoyl)aminopropylamine(-ArN3) attached to the terminal phosphate (reagents I, II, and III, respectively) was studied at 37 degrees C. It was established that during irradiation the reagents are inactivated, loosing their affinity to the target. A kinetic equation describing the modification was suggested. From the dependence of the time-limited modification level on the reagent concentration, the association constants of the reagents with the target were determined: [Kx = (9.9 +/- 0.4) x 10(4), (1.1 +/- 0.1) x 10(5), and (8.4 +/- 2.1) x 10(6) M-1 for reagents I, II, and III, respectively] and the efficiency of the modification in the complex gamma ef (ca. 0.3 for all the reagents) were determined. From the dependence of the modification level [PZ]/p0 on time for reagent II, the rate constant was determined for the rate-determining step of the photomodification k0 = (7.9 +/- 0.9) x 10(-3) s-1, which is close to the rate constant for the photolysis of p-azidotetrafluorobenzoic acid kp = (5.5 +/- 0.3) x 10(-3) s-1.     

Human dentine as a hydrogel

The pores (tubules) of human dentine in 0.02-cm planoparallel sections of newly extracted permanent teeth were investigated. By the conventional scanning electron microscopy these pores appear empty, but by the newly developed scanning-probe microscopy the presence of a complex matrix could be established. By measuring the transport of neutral myoglobin by diffusion alone and diffusion+bulk flow, the area of dentine occupied by the matrix was calculated to be 1.9 +/- 0.9% and 2.3 +/- 0.5%, respectively. The hydraulic conductivity was surprisingly small, 1.35 +/- 0.55 x 10(-7) ml/(s.cm2 dentine) at a pressure difference of 0.1 kPa across a 1-cm thick section. This suggests a hydrogel with a relatively dense network, the width of meshes estimated at 2 x 30 nm. In line with this concept, enzymatic degradation of the organic matter increased the hydraulic conductivity 3000 times. By studying the transport of negatively charged myoglobin, the matrix was calculated to carry 18 mEq/l of positive charges. Due to the consequent attraction of small, negative ions and thence of water, the pressure within the matrix would be about 1.33 kPa, a force which will act to immobilize the water in the channels. The concept of a hydrogel in the dentine tubules was also supported by the finding that shielding the charges with bathing media of high ionic strength reduced the hydraulic conductivity.     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the initial acceptor in cytochrome c oxidase

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.