The effects of magnesium on hydroxyapatite formation in vitro from CaHPO4 and Ca4(PO4)2O at 37.4 degrees C

The effects of magnesium ion on the formation of calcium-deficient hydroxyapatite [Ca9HPO4(PO4)5OH, CDHAp] from CaHPO4 and Ca4(PO4)2O dissolution were investigated using two magnesium sources: Mg3(PO4)2 (chemical system 1) or MgCl2 . H2O (chemical system 2) solutions. Because chloroapatite does not form from aqueous solutions, the use of two magnesium sources facilitated the determination of magnesium's role during synthetic hydroxyapatite formation in vitro and possible related effects during biomineralization. Isothermal calorimetry determined the progress of reactions. Two peaks are observed as heat is evolved during the formation of CDHAp in water at 37.4 degrees C. The nucleation and growth of CDHAp are the corresponding mechanisms. Although the time for complete reaction and total heat-of-reaction DeltaHr remain constant, the height of the first peak is reduced as the concentration of magnesium ion approaches 4 mM in either chemical system. Magnesium does not substitute into CDHAp even though there are calcium vacancies available. Subsequent increases cause the remaining heat peak to broaden and the time required for complete reaction to approach 24 hours as the initial MgCl2 concentration reaches 100 mM. Supersaturation limits chemical system 1 to Mg3(PO4)2 concentrations below 10 mM. A MgCl2 concentration of 3.16 M precludes CDHAp from forming for over 3 months; rather newberyite, MgHPO4 . 3H2O, precipitates. The morphology and surface area of the CDHAp formed in 100 mM MgCl2 solution are comparable to those of CDHAp formed in water. The surface areas are approximately 80 m2/g. Magnesium concentrations below 4 mM only inhibit nucleation whereas those above 4 mM inhibit growth as well. Magnesium phosphate complexes are more inhibitory than magnesium chloride complexes. Increasing the liquid-to-solids ratio or agitation significantly increases the induction period before reaction initiates. Increasing the liquid-to-solids ratio increases the time span for growth whereas increasing agitation decreases the time span for growth. The large inhibitory effect of agitation suggests quiescent systems are more suitable for determining the kinetics of HAp formation. A magnesium inorganic chemical activity (alphaMg = gammaMg[Mg2+]) many times greater than that in biological fluids is required before inhibition of hydroxyapatite formation is realized.     

Importance of intrinsic calf vasodilator capacity in determining distribution of skeletal muscle perfusion during supine bicycle exercise in patients with left ventricular dysfunction

Although there is interest in forming synthetic analogs of hard tissues at physiologic temperature, significant gaps in knowledge exist with respect to the mechanisms by which precursor solids convert to apatites and also with respect to the apatite compositions that may be formed. In this study calcium-deficient HAp [Ca9(HPO4)(PO4)5OH] was prepared by hydrolysis of tricalcium phosphate (TCP), alpha-Ca3(PO4)2. The kinetics of HAp formation were studied as a function of temperature by isothermal calorimetry. TCP hydrolyzed completely within about 12 h, and the hydrolysis reaction evolved 133 kJ/mol of HAp formed. Although the kinetics of hydrolysis exhibited a strong temperature dependence, the mechanistic path taken appeared independent of temperature. The fluoridation of hydroxyapatite compositions having Ca/P ratios higher than 1.59 previously has been investigated. However, little work has been done on the fluoridation of more calcium-deficient hydroxyapatite. Ca9(HPO4)(PO4)5OH was formed at temperatures between 37.4 degrees and 55 degrees C to vary its morphology. These preparations then were reacted in NaF solution and the kinetics of fluoride incorporation studied. Solution chemical analyses were used to determine the amounts of fluoride incorporated. The extent of hydroxyl replacement by fluoride ranged from 17 to 72% and correlated with the surface area of the parent HAp.     

Hypoxia, temperature, and pH/CO2 effects on respiratory discharge from a turtle brain stem preparation

An in vitro brain stem preparation from adult turtles (Chrysemys picta) was used to examine the effects of anoxia and increased temperature and pH/CO2 on respiration-related motor output. At pH approximately 7.45, hypoglossal (XII) nerve roots produced patterns of rhythmic bursts (peaks) of discharge (O.74 +/- 0.07 peaks/min 10.0 +/- 0.6 s duration) that were quantitatively similar to literature reports of respiratory activity in conscious, vagotomized turtles. Respiratory discharge was stable for 6 h at 22 degrees C; at 32 degrees C, peak amplitude and frequency progressively and reversibly decreased with time. Two hours of hypoxia had no effect on respiratory discharge. Acutely increasing bath temperature from 22 to 32 degrees C decreased episode and peak duration and increased peak frequency. Changes in pH/CO2 increased peak frequency from zero at pH 8.00-8.10 to maxima of 0.81 +/- 0.01 and 1.44 +/- 0.02 peaks/min at 22 degrees C (pH 7.32) and 32 degrees C (pH 7.46), respectively; pH/CO2 sensitivity was similar at both temperatures. We conclude that 1) insensitivity to hypoxia indicates that rhythmic discharge does not reflect gasping behavior, 2) increased temperature alters respiratory discharge, and 3) central pH/CO2 sensitivity is unaffected by temperature in this preparation (i.e., Q10 approximately 1.0).     

Denaturation and association of DNA sequences by certain polypropylene surfaces

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule of 75 kDa per subunit with one molecule of cofactor NAD per dimer. It undergoes unfolding and complete dissociation in presence of 8 M urea at pH 7.0 by 10 min. It can be functionally reconstituted almost quantitatively in 2 h by dilution with 20 mM sodium phosphate buffer, pH 7 containing 1 mM extraneous NAD under a second order kinetics [Bhattacharyya, D. (1993) Biochemistry 32, 9726-9734]. Denaturation between 10-60 min inversely affects both the rate and maximum recovery of activity upon refolding. Aggregation of this protein has not been observed under these conditions. The time dependent reaction at the unfolded state is independent of pH between 5.4-10.4 but strongly dependent on temperature of denaturation between 0-20 degrees C. Unfolding at 0 degrees C divides the protein largely into two populations-34% of fast folding species following an apparent first order kinetics and 59% of slow folding species following a second order kinetics of reactivation. A very fast folding species of low abundance 3.5-7.5% depending on temperature of denaturation has been identified, which gets active status within the dead time of mixing. Interaction with the active site directed fluorescence probe 1-anilino 8-naphthalene sulfonic acid (1-ANS) and estimation of bound NAD suggest that the catalytic region of this enzyme is not formed in the long term denatured samples. The whole process of reactivation is catalysed by peptidyl prolyl cis-trans isomerase and thus suggests that one or more proline residues stereochemically control the rate limiting step of reactivation.     

Preparation of high-temperature stabilized beta-tricalcium phosphate by heating deficient hydroxyapatite with Na4P2O7 x 10H2O addition

In this paper, the high-temperature stabilized beta-tricalcium phosphate (betaTCP, beta-Ca3(PO4)2) were prepared by heating the deficient HAP (d-HAP, Ca10-x(HPO4)x(PO4)6-x(OH)2-x) with tetra-sodium diphosphate decahydrate (NP, Na4P2O7 x 10H2O) addition. The betaTCP, d-HAP and d-HAP doped with 2.5, 5, 7.5 and 10 wt % NP were heated to different temperatures and were investigated by X-ray diffraction analysis (XRD) and Fourier-transformed infrared spectroscopy (FTIR). The results demonstrated that the HPO4(2-) of d-HAP condensed into P2O7(4-) occurred before 650 degrees C. The P2O7(4-) ions could be traced in the FTIR spectrum when the d-HAP was heated up to 750 degrees C. The reaction of P2O7(4-) with OH- did not occur instantly but over a wide range of temperatures. The d-HAP doped with NP would decrease the decomposition temperature of d-HAP. NP doped into d-HAP not only induced the d-HAP decomposition at lower temperature but also stabilized the betaTCP crystal structure at higher-temperature. It could also increase the conversion temperature of betaTCP to alphaTCP from 1180 degrees C up to 1300 degrees C. We could successfully prepare high-temperature (up to 1300 C) stabilized ffTCP by heating NP doped d-HAP.     

Comparison of brain tissue metabolic changes during ischemia at 35 degrees and 18 degrees C

BACKGROUND: We evaluated brain tissue oxygen pressure (PO2), carbon dioxide pressure (PCO2) and pH during ischemia with brain temperature at 35 degrees and 18 degrees C in the same patient. METHODS: Surgery was performed in a 60-year-old woman to clip a large aneurysm in the left internal carotid artery (ICA). A Paratrend 7 probe measuring PO2, PCO2, and pH was inserted into tissue at risk for ischemia during ICA occlusion and brain protection was provided with 9% desflurane. One week later, hypothermic circulatory arrest with brain temperature at 18 degrees C was performed for aneurysm clipping and tissue measurements were obtained during ischemia and rewarming. RESULTS: At 35 degrees C, ICA occlusion for 16 minutes produced tissue hypoxia (PO2 = 0) and acidosis (pH = 6.70). The rate of increase of hydrogen ion (H+) reached 50 nEq.L(-1).min(-1) during ICA occlusion and there was a slow recovery of acidosis at the end of the ischemic period. During hypothermic circulatory arrest, tissue PO2 was sensitive to decreases in blood pressure and decreased rapidly during exsanguination. Although tissue pH decreased to 6.5 with 30 min of no pump flow, the rate of H+ increase during hypothermic arrest was one-third of that seen during ischemia at 35 degrees C. During rewarming from profound hypothermia, two phases of recovery from acidosis were observed, one during CO2 clearance and one after tissue reoxygenation. Recovery of acidosis occurred sooner at 18 degrees C than at 35 degrees C. CONCLUSIONS: These results show that tissue acidosis develops more slowly and recovers more rapidly with hypothermic ischemia. This may be an important mechanism of reduced ischemic injury during hypothermia.     

Physiopathology of bedsores

Listeria monocytogenes is a food-borne pathogenic bacterium that can be found in soft cheese. At the beginning of cheese ripening, the pH is about 4.85-4.90. The aim of this work was to study the influence of temperature, preincubation temperature (temperature at which the inoculum was cultivated) and initial bacterial concentration on the survival of L. monocytogenes (strain Scott A) at pH 4.8. It was demonstrated in an earlier study that these factors did influence growth kinetics. Survival studies of L. monocytogenes were done in a laboratory broth simulating cheese composition. Four test temperatures (2, 6, 10 and 14 degrees C) and two preincubation temperatures were studied (30 degrees C or the test temperature). Listeria monocytogenes (strain Scott A) was unable to grow at pH 4.8 under all conditions tested. The time for 10% survival was about 11 and 2 d, at 2 degrees C with preincubation at 2 degrees C and 30 degrees C, respectively; 9 d at 6 degrees C with preincubation at 6 degrees C; 4 d at 6 degrees C with preincubation at 30 degrees C; and 1 d at 14 degrees C with preincubation at 14 degrees C or at 30 degrees C. The results show that survival of L. monocytogenes (strain Scott A) at pH 4.8 is not dependent on initial bacterial concentration but on both the test and preincubation temperatures.     

Effects of GABAB receptor antagonism on the development of pentylenetetrazol-induced kindling in mice

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular beta-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 degrees C and 30 degrees C and slightly less at 35 degrees C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 degrees C) during an incubation period of 5 days. Supernatants of cultures grown at 25 degrees C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4-5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 degrees C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 degrees C and 30 degrees C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin.     

Adsorptive Removal of Parts per Million Level Selenate Using Iron-Coated GAC Adsorbents

Selenate removal by adsorption using iron-coated granular activated carbons (Fe-GACs) is reported in this study. Adsorption kinetics and equilibrium experiments with initial selenium concentration of 1 mg/L were conducted under three different ionic strengths to study selenate adsorption behavior. Selenate adsorption reached equilibrium within 48 h with more than 85% of the equilibrium capacities being obtained within the first 6 h. High removal efficiency (i.e., >75%) was achieved for pH range of 2–5. Acid-base titration experiments showed point of zero charge (pHpzc) at pH 7.5 for the tested Fe-GAC. Pseudo-second-order kinetic model characterized selenate adsorption kinetics well (R2 = 0.999) and the rate constant decreased with ionic strength. Adsorption capacity decreased significantly with increasing ionic strength, which was not observed in selenite adsorption with the same adsorbent. Competitive adsorption with other four oxyanions (SiO32?, SO42?, PO43?, and CO32?) showed that selenate removal efficiency was reduced to various degrees in the presence of each individual anion. Competitive adsorption of binary adsorbates (selenite and selenate) showed a decreasing trend of selenite adsorption capacity with decreasing ionic strength, indicating stronger competition of selenate against selenite under the low ionic strengths. The Sheindorf-Rebuhn-Sheintuch multiadsorbate competitive adsorption model was applied to quantify the binary competitive adsorption between selenate and selenite.     

Right-shifting the oxyhemoglobin dissociation curve with RSR13: effects on high-energy phosphates and myocardial recovery after low-flow ischemia

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 degrees C for production of mesenterocin 52A and 25 degrees C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth.     

Studies of the reaction of acetaldehyde with deoxynucleosides

The reaction of acetaldehyde with deoxynucleosides was studied in buffered solutions at room temperature (22-24 degrees C) and neutral pH. Reaction products were obtained with all deoxynucleosides with the exception of thymidine, as shown by reversed-phase HPLC analysis. The order of reactivity was dGuo > dAdo > dCyd, for which three, two and one reaction products, respectively, were obtained. We report here data on the kinetics of the reactions, the stability of the adducts at physiological pH, product yields, UV-spectroscopic data at different pH values, and describe the synthesis, isolation and structural characterization by FAB/MS and NMR of the stable adducts of acetaldehyde with dGuo. Furthermore, the formation of adducts with dGuo by the cooperative reaction of Aa with ethanol was studied.     

Micellar electrokinetic capillary chromatography (MECC) of UV-absorbing constituents in normal urine: a chemometric optimisation of the separation

A micellar electrokinetic capillary chromatography (MECC) method when compared to free solution capillary electrophoresis (CZE) was shown to offer improved selectivity and resolution for the separation of UV-absorbing components of human urine. Some of the factors affecting MECC separation e.g. methanol concentration, sodium dodecyl sulphate (SDS) concentration, beta-cyclodextrin (beta-CD) concentration, voltage, pH, temperature and electrolyte additives (urea, beta-CD and Brij 35) were optimised using chemometric techniques. Three-level three-factor (3(3)) factorial designs and simplex optimisation were used to achieve optimised conditions with the goal of obtaining the maximum number of peaks in the shortest possible analysis time. Using a TSP CE2000 instrument with detection from 195-300 nm and fitted with a 75 microns x 44 cm (37 cm effective length) fused silica capillary the final optimum conditions were found to be, an electrolyte consisting of 30 mM sodium tetraborate, pH 10, containing 75 mM SDS and 10 mM beta-CD, 15 degrees C, 20 kV, 4 s hydrodynamic injection of filtered urine. These conditions were capable of separating 70 peaks from a normal human urine pool in less than 12 min. The separation of components in urine using the optimised MECC was simpler, more reproducible, faster and gave better resolution than gradient reversed-phase high performance liquid chromatography.     

Effect of salts on the solubility of thermolysin: a remarkable increase in the solubility as well as the activity by the addition of salts without aggregation or dispersion of thermolysin

Thermolysin is remarkably activated in the presence of high concentrations (1-5 M) of neutral salts [Inouye, K. (1992) J. Biochem. 112, 335-340]. The activity is enhanced 13-15 times with 4 M NaCl at pH 7.0 and 25 degrees C. In this study, the effect of neutral salts on the solubility of thermolysin has been examined. Although the solubility was only 1.0-1.2 mg/ml in 40 mM Tris-HCl buffer, pH 7.5, in the temperature range between 0 and 60 degrees C, it was increased greatly by the addition of salts. With NaCl, the solubility showed a bell-shaped behavior with increasing NaCl concentration, and the maximum solubility (10 mg/ml) was at 2.0-2.5 M NaCl. With LiCl and NaI, it increased progressively to 20-50 mg/ml with increasing salt concentration up to 5 M. The solubility observed in the presence of salts decreased with increasing temperature from 0 to 60 degrees C, and also with the order of chaotropic anion effect. The molecular weight of thermolysin was estimated to be 33.0(+/-2.5) x 10(3) in the presence of 0-3 M NaCl, suggesting that thermolysin exists as a monomer in the presence or absence of 3 M NaCl. The possibility that aggregation and/or dispersion of thermolysin might be related to the remarkable activation by salt was ruled out.     

Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.     

Electrophoretic separation of recombinant tissue-type plasminogen activator glycoforms: validation issues for capillary isoelectric focusing methods

Attempts were made to validate a capillary isoelectric focusing (cIEF) method for a recombinant glycoprotein as an alternative technique to slab gel isoelectric focusing methods routinely used to monitor such charge heterogeneity. The cIEF method principally separates the charged glycoforms of recombinant tissue-type plasminogen activator (rt-PA) on the basis of their sialic acid content. Nine to ten distinct peaks were consistently resolved, with the profile dependent on the class of ampholyte used. The pI of rt-PA measured with synthetic pI standards was in the range pH 6.5-7.5 with the migration of the standards affected by the presence of the protein. The method showed an acceptable recovery of > 100% and had good sensitivity where 25 ng of protein could be resolved into constituent peaks. Recovery of both major peaks and total protein measured by peak areas was linear over a wide range from 50-1000 micrograms/mL. A detailed study showed that when a capillary had been used for some time, capillary age affected peak migration times and, to a lesser extent, resolution. Peak migration times were stable over a temperature range of 15-30 degrees C, and decreased predictably with increasing voltages (400-600 V/cm) and decreasing N,N,N',N'-tetramethylethylene diamine (TEMED) concentrations (0.4-1.5% v/v). Overall the data indicated that this methodology has the potential to be used in the commercial release of protein pharmaceuticals if variability resulting from capillary age and lot were resolved. Even in its present format the method equals the performance of slab gel IEF whilst offering significant improvements in ease of operation and in time and reagent use.     

ATP-Induced phosphorylation of the sarcoplasmic reticulum Ca2+ ATPase: molecular interpretation of infrared difference spectra

Time-resolved infrared difference spectra of the ATP-induced phosphorylation of the sarcoplasmic reticulum Ca2+-ATPase have been recorded in H2O and 2H2O at pH 7.0 and 1 degrees C. The reaction was induced by ATP release from P3-1-(2-nitro)phenylethyladenosine 5'-triphosphate (caged ATP) and from [gamma-18O3]caged ATP. A band at 1546 cm-1, not observed with the deuterated enzyme, can be assigned to the amide II mode of the protein backbone and indicates that a conformational change associated with ATPase phosphorylation takes place after ATP binding. This is also indicated between 1700 and 1610 cm-1, where bandshifts of up to 10 cm-1 observed upon protein deuteration suggest that amide I modes of the protein backbone dominate the difference spectrum. From the band positions it is deduced that alpha-helical, beta-sheet, and probably beta-turn structures are affected in the phosphorylation reaction. Model spectra of acetyl phosphate, acetate, ATP, and ADP suggest the tentative assignment of some of the bands of the phosphorylation spectrum to the molecular groups of ATP and Asp351, which participate directly in the phosphate transfer reaction: a positive band at 1719 cm-1 to the C==O group of aspartyl phosphate, a negative band at 1239 cm-1 to the nuas(PO2-) modes of the bound ATP molecule, and a positive band at 1131 cm-1 to the nuas(PO32-) mode of the phosphoenzyme phosphate group, the latter assignment being supported by the band's sensitivity toward isotopic substitution in the gamma-phosphate of ATP. Band positions and shapes of these bands indicate that the alpha- and/or beta-phosphate(s) of the bound ATP molecule become partly dehydrated when ATP binds to the ATPase, that the phosphoenzyme phosphate group is unprotonated at pH 7.0, and that the C==O group of aspartyl phosphate does not interact with bulk water. The Ca2+ binding sites seem to be largely undisturbed by the phosphorylation reaction, and a functional role of the side chains of Asn, Gln, and Arg residues was not detected.     

Plasma thyroid hormone kinetics are altered in iron-deficient rats

Iron deficiency anemia is associated with lower plasma thyroid hormone concentrations in rodents and, in some studies, in humans. The objective of this project was to determine if plasma triiodothyronine (T3) and thyroxine (T4) kinetics were affected by iron deficiency. Studies were done at a near-thermoneutral temperature (30 degrees C), and a cool environmental temperature (15 degrees C), to determine plasma T3 and T4 kinetics as a function of dietary iron intake and environmental need for the hormones. Weanling male Sprague-Dawley rats were fed either a low Fe diet [iron-deficient group (ID), <5 microg/g Fe] or a control diet [control group (CN), 35 microg/g Fe] at each temperature for 7 wk before the tracer kinetic studies. An additional ID group receiving exogenous thyroid hormone replacement was also used at the cooler temperature. For T4, the disposal rate was >60% lower (89 +/- 6 vs. 256 +/- 53 pmol/h, P < 0.001) in ID rats than in controls at 30 degrees C, and approximately 40% lower (192 +/- 27 vs. 372 +/- 26 pmol/h, P < 0.01) in ID rats at 15 degrees C. Exogenous T4 replacement in a cohort of ID rats at 15 degrees C normalized the T4 concentration and the disposal rate. For T3, the disposal rate was significantly lower in ID rats in a cool environment (92 +/- 11 vs. 129 +/- 11 pmol/h, P < 0.01); thyroxine replacement again normalized the T3 disposal rate (126 +/- 12 pmol/h). Neither liver nor brown fat thyroxine 5'-deiodinase activities were sufficiently different to explain the lower T3 disposal rates in iron deficiency. Thus, plasma thyroid hormone kinetics in iron deficiency anemia are corrected by simply providing more thyroxine. This suggests a central regulatory defect as the primary lesion and not peripheral alterations.     

Synthesis of zwitterionic acrylic acid buffers for isoelectric focusing in immobilized pH gradients

A range of zwitterionic acrylic acid derivatives, buffering in the neutral and basic pH ranges, have been synthesized by the Mannich reaction of malonic acid, formaldehyde and a secondary amine. These compounds include 2-(4-morpholinomethyl)propenoic acid pK2 7.59 +/- 0.03 (23 degrees C), 2-[bis(2-hydroxyethyl)aminomethyl]propenoic acid pK2 approximately 8.6 (20 degrees C), 2-[bis(2-hydroxypropyl) aminomethyl]propenoic acid PK2 approximately 8.7 (20 degrees C), 2-[N-(2-hydroxyethyl)-N-methylaminomethyl]propenoic acid pK2 9.22+/-0.08 (22 degrees C), 2-[N-ethyl-N-(2hydroxyethyl)aminomethyl]-propenoic acid pK2 approximately 9.6 (20 degrees C), and 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid, which has a sigmoidal buffering profile over the pH range 3-10. These zwitterionic acrylic acid buffers were successfully copolymerized with acrylamide to prepare immobilized pH gradients (IPGs) in the neutral to alkaline portion of the pH range. Bovine erythrocyte carbonic anhydrase isozymes were resolved on a pH 5-8 IPG prepared using 2-[4-(2-carboxyprop-2-enyl)piperazinylmethyl]propenoic acid as the immobilized buffer, and horse heart myoglobin was focused on pH 7.1-8.1 and pH 7.5-7.7 IPGs, using 2-(4-morpholinomethyl)propenoic acid as the immobilized buffer. In both cases the pK 9.3 Immobiline compound was used as the strongly basic titrant. These new compounds, besides possessing more hydrophilic residues than the corresponding commercial basic acrylamido buffers (Immobilines), resist hydrolysis at alkaline pH values.     

The effect of the elemental sulfur reaction product on the leaching of galena in ferric chloride media

The dissolution of galena in 0.3 M FeCl₃-0.3 M HC1 solutions containing 0 to 6 M LiCl was studied at 80 °C, and parabolic kinetics were observed at all LiCl concentrations. The leaching rate increases gradually with increasing LiCl concentrations to ≈4 M LiCl; the presence of >4 M LiCl results in a rapid increase in the leaching rate. The solubility of PbCl₂ in 0.3 M FeCl₃-0.3 M HC1 solutions containing 0 to 6.5 M LiCl was measured over the temperature range of 50 °C to 90 °C. The solubility increases systematically with increasing temperature and LiCl concentration. The parabolic kinetics, coupled with the correlation between the leaching rate and the solubility of PbCl₂, suggest that the dissolution of galena is controlled by the outward diffusion of the PbCl₂ reaction product through the constantly thickening layer of elemental sulfur formed during leaching. This conclusion is also supported by various morphological studies which consistently indicated a thin layer of PbCl₂ between the corroding galena and the porous elemental sulfur reaction product.     

Genistein exerts estrogen-like effects in male mouse reproductive tract

The 'retention analysis method', which is based on size-exclusion chromatography (SEC) in conjunction with an arsenic-specific detector (graphite furnace atomic absorption spectrometer, GFAAS), was used to study the effect of pH (range 2.0-10.0), temperature (4, 25 and 37 degrees C), and the concentration of glutathione in the mobile phase (0.5-7.5 mM) on the formation of arsenic-glutathione species after injection of sodium arsenite using phosphate-buffered saline solutions as mobile phases. The formation of arsenic-GSH species was facilitated by low temperatures (4 degrees C), pH 6.0-8.0 and high concentrations of glutathione (7.5 mM) in the mobile phase. Simulating the physicochemical parameters found inside human red blood cells (approximately 3.0 mM glutathione, 37 degrees C, pH 7.4) and hepatocytes (approximately 7.5 mM glutathione, 37 degrees C, pH 7.4), SEC-GFAAS provided evidence for the formation of arsenic-glutathione species under these conditions. In addition, the 'chelating agent', sodium DL-2,3-dimercapto- -propanesulfonate (1.0 and 2.0 mM) was demonstrated to bind arsenous acid stronger in the presence of glutathione (7.5 mM) under these conditions (PBS buffer, pH 7.4, 37 degrees C).