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Biomedical subjects

R E Weber

Publications and source records attributed to R E Weber.

At least 19 recordsLinked to original sources
PubMed1992

Use of ionic and zwitterionic (Tris/BisTris and HEPES) buffers in studies on hemoglobin function.

The functional characteristics of hemoglobin (Hb) depend on oxygenation-linked proton and anion binding and thus on solvent buffer groups and ionic composition. This study compares the oxygenation properties of human Hb in ionic [tris(hydroxymethyl)aminomethane (Tris) and BisTris] buffers with those in zwitterionic N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer under strictly controlled chloride concentrations at different pH values, two temperatures, and in the absence and presence of the erythrocytic cofactor, 2,3-diphosphoglycerate (DPG). In contrast to earlier studies (carried out at the same or different chloride concentrations) it shows only small buffer effects that are manifested at low chloride concentration and high pH. These observations suggest chloride binding to the Tris buffers, which reduces the interaction with specific chloride binding sites in the Hb. The findings indicate that HEPES allows for more accurate assessment of Hb-oxygen affinity and its anion and temperature sensitivities than ionic buffers and advocates standard use of HEPES in studies on Hb function. Precise oxygen affinities of Hb dissolved in both buffers are defined under standard conditions.

Binding Sites↗
PubMed1991

Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering.

Of two closely related species of geese, one, the greylag goose, lives in the Indian plains all year round, while the other, the bar-headed goose, lives at the Tibetan lakes and migrates across the Himalayas to winter in India. Another species, the Andean goose, lives in the High Andes all year round. Possession of a Hb with high oxygen affinity helps to adapt bar-headed and Andean geese to high altitudes. The Hb amino acid sequences of the bar-headed and the greylag geese differ by four substitutions, of which only one is unique among bird sequences: Pro-119 alpha (H2)----Ala. Perutz proposed that the two-carbon gap left by this substitution at the alpha 1 beta 1 contact raises the oxygen affinity, because it relaxes the tension in the deoxy or T structure [Perutz, M. F. (1983) Mol. Biol. Evol. 1, 1-28]. It was later found that the Hb of the Andean goose has a gap in the same position, due to the complementary substitution Leu-55 beta (D6)----Ser. We have tested Perutz's hypothesis by introducing each of these substitutions into human globin synthesized in Escherichia coli. The reconstituted Hbs combine cooperatively with oxygen. Their oxygen affinities exceed that of normal human Hb by an even larger factor than that found between the high-flying geese and the greylag goose. The mutant Hb Met-55 beta (D6)----Ser was crystallized. Its structure is the same as that of HbA, except in the immediate environment of the gap left by the substitution of the serine for the methionine side chain, which evidently causes the increased oxygen affinity of this Hb.

Adaptation, Physiological↗
PubMed1991

Respiratory impairment in crustaceans and molluscs due to exposure to heavy metals.

1. We have assessed, using current literature, the respiratory consequences of water-borne heavy metal exposure in crustaceans and molluscs. 2. We suggest that in lethal and sub-lethal concentrations the essential metals Cu and Zn act on the respiratory system primarily by disrupting gill function which results in the development of internal hypoxia, although reparation can be accomplished even at "high" sub-lethal concentrations. 3. The more toxic xenobiotes such as Hg (and perhaps to a lesser extent Cd) may interfere with the respiratory system at every level of organisation including cellular respiration itself.

Animals↗
PubMed1991

Primary structure and oxygen-binding properties of the hemoglobin from the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta). Evidence for phylogenetic isolation.

The primary structures of the alpha- and beta-hemoglobin chains of the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta) are presented. Chain separation was performed by carboxymethyl-cellulose chromatography. The peptides, obtained by tryptic digestion of the oxidized chains, were prefractionated by gel chromatography and isolated by reversed-phase HPLC. For sequence analysis gas and liquid phase sequencers were employed. The tenrec hemoglobin consists of one alpha- and two beta-chains the latter occurring in a 1:1 ratio and differing in beta 16 Gly/Cys and beta 118 Phe/Leu. Two external cysteine residues at beta 16 and beta 52 cause reversible polymerization to octamers and most likely irreversible formation of higher polymers. A comparison of the whole chains and certain positions of tenrec hemoglobin with those of Insectivora sensu strictu, Scandentia and Proto- and Metatheria corroborates a long and independent evolution of tenrec and its phylogenetic isolation from the Insectivora s.str. (hedgehog, musk shrew and mole). Replacements at positions involved in heme and subunit interface contacts are discussed. Compared to human hemoglobin the tenrec pigment shows a low intrinsic oxygen affinity as well as lower chloride and temperature sensitivities, a reduced Bohr effect and a strong response to 2,3-DPG. The possible adaptive significance of these properties is discussed in relation to the large diurnal body temperature variations seen in tenrecs.

Amino Acid Sequence↗
PubMed1990

Crystallization of the rainbow trout (Salmo gairdneri) haemoglobin IV.

Crystals of rainbow trout (Salmo gairdneri) haemoglobin IV were grown in mini batches from a solution of ammonium sulphate. Large single crystals grew over five days and were up to 2 mm in length. X-ray diffraction experiments indicated a space group of C222(1) with unit cell dimensions of a = 85.3 A, b = 94.6 A and c = 105.7 A. The crystals diffract to better than 2.5 A but exhibit some mosaicity along the c axis.

Animals↗
PubMed1989

Gas exchange in fish gills with parallel inhomogeneities.

Using a mathematical model, the effects of water and blood flow inequality on gas exchange in the fish gill were investigated. The results show that O2 exchange is more sensitive to flow inequalities than CO2 exchange. Oxygen exchange is most sensitive to blood flow inequality, whereas carbon dioxide exchange is equally sensitive to inequalities in water and blood flow. Furthermore, it is shown that the effects of flow inequalities cannot be abolished by matching the two flows exactly. The results suggest that the greatest potential for regulation of gas exchange lies on the blood side of the exchanger, and that adjustments in flow distributions to maintain homeostasis in oxygen exchange in the face of changing environmental conditions and/or changing organismic demands, most likely will be accompanied by a change in the acid-base status of the animal.

Animals↗
PubMed1989

Deposition of cigarette smoke particles in the rat.

The fractional deposition of cigarette smoke particles in the respiratory tracts of rats was studied. Male and female rats were conditioned in nose-only exposure tubes 25 min/day for 2 days, exposed to cigarette smoke at mass concentrations of 95 or 341 mg/m3, 25 min/day for 3 days, and then exposed to smoke at mass concentrations of 212 and 657 mg/m3, 25 min/day for 5 days. Mainstream cigarette smoke was generated by a modified Walton smoking machine from two 1R3 research cigarettes burned sequentially for each exposure. Deposition studies were conducted by placing the rats in plethysmograph tubes to allow respiratory minute volume measurements during exposure, then exposing them to [14C]dotriacontane-labeled cigarette smoke at mass concentrations of 202 or 624 mg/m3 for 25 min, using the same smoking machine. Size distribution, real-time concentration, and 14C activity of the smoke particles were determined using a multijet Mercer impactor, a real-time aerosol monitor, and filter samples, respectively. Immediately after the exposure, the rats were terminated to determine the distribution of the 14C. Individual lung lobes, trachea and lobar bronchi, head, larynx, kidneys, liver, gastrointestinal (GI) tract, blood, and depleted carcass of each rat were analyzed for 14C content. Results showed that the GI tract contained 16-31% of the total activity, indicating significant clearance from the large airways and nose to the GI tract during the exposure and during the 10-15 min between the cessation of the exposure and the removal of the organs. Total deposition of the inhaled 14C activity was 20.1 +/- 1.6% for both exposure concentrations. The intrapulmonary deposition fractions (lung lobes plus airways below the lobar bronchi) were 12.4 +/- 0.9 and 15.9 +/- 1.4% for concentrations of 202 and 624 mg/m3, respectively, suggesting a slight enhancement in upper airway deposition for animals exposed to the higher smoke concentration.

Alkanes↗
PubMed1989

Continuous cardiac output determination using transtracheal Doppler: initial results in humans.

Transtracheal Doppler, a procedure developed in the authors' laboratory, provides an estimate of cardiac output in endotracheally intubated dogs. The present study reports initial results in humans with a Doppler probe incorporated into an endotracheal tube. The system was first calibrated by empirically determining phi, the angle of the ultrasound beam with respect to the blood velocity vector. For a best least squares fit for phi, aortic diameters can be calibrated over a range of 22-37 mm. The calibration was then tested in a separate group of patients using the same empirically derived angle to calculate cardiac outputs. The transtracheal Doppler cardiac outputs show good correlation with those obtained by thermodilution over a range of cardiac outputs, 2.69-8.62 1/min, R2 = 0.835.

Adolescent↗
PubMed1989

Amino-acid sequences and functional differentiation of hemoglobins A and D from swift (Apus apus, Apodiformes).

The blood of the adult swift contains one major (HbA = alpha 2A beta 2) and two minor components (HbD = alpha 2D beta 2 and HbD'). The components were separated by FPLC with a TSK SP-5 PW-column in phosphate buffers, and were eluted with a linear NaCl gradient. HbD' could be detected only in freshly prepared hemolysates with the sensitive FPLC separation method. The globin chains were separated on a cation exchanger (CM-cellulose), the tryptic peptides by HPLC with a RP-2 LiChrosorb column. Their amino-acid sequences were determined by automatic Edman degradation with the film- or gas-phase method. For the alpha A-, alpha D- and beta-chains, peptide alignment was achieved by homologous comparison with the corresponding chains of the greylag goose (Anser anser). The structural significance of the substitutions was examined with the aid of molecular graphics. The oxygen-binding properties of the stripped hemolysate and of HbA and HbD and their dependence on pH, temperature and inositol polyphosphate are presented and discussed with reference to molecular structures and hypothermy that occurs during torpidity.

Amino Acid Sequence↗
PubMed1989

High-altitude respiration of falconiformes. The primary structures and functional properties of the major and minor hemoglobin components of the adult White-Headed Vulture (Trigonoceps occipitalis, Aegypiinae).

The primary structures of the hemoglobin components Hb A and Hb D of White-Headed Vulture (Trigonoceps occipitalis) are presented. The globin chains were separated on CM-Cellulose in 8M urea buffer, the components by FPLC in phosphate buffers. The amino-acid sequences were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid phase and gas-phase sequenators. The sequences differ from those of European Black Vulture by only one mutation in the alpha A-chains (alpha 137). The alpha D-chains and the beta-chains are identical. This means that for the first time identical minor components in birds have been found. An updated list of identical globin chains is presented. Hb D exhibited a higher oxygen affinity than Hb A. At pH 7.5 and 38 degrees C P50 values of 0.80 and 0.64 kPa (6.0 and 4.8 mm Hg), respectively. Both hemoglobins showed similar Bohr factors displayed a pronounced sensitivity to inositol hexakis(phosphate), which increased P50 values of Hbs A and D to 4.0 and 3.6 kPa (30 and 26 mm Hg), respectively. The molecular and physiological significance of the findings is discussed with special reference to oxygen transport by hemoglobin at high altitude.

Altitude↗
PubMed1989

Allosteric transition in hemoglobin (alpha 2A beta 2I) from the rhynchocephalian reptile relict Sphenodon punctatus.

The major hemoglobin component Hb A of the tuatara, Sphenodon punctatus, a relict of the rhynochocephalian reptiles that lived 200 million years ago, was investigated in the light of the apparent contradiction inherent in an effect of organic phosphate cofactors on the oxygen affinity of hemoglobins exhibiting hyperbolic oxygen equilibrium curves. The heterotropic allosteric effect of ATP, the major erythrocytic cofactor in the tuatara, is shown to be correlated with distinct homotropic interactions (Hill's cooperativity coefficient at half-saturation, n50, attaining 1.3-1.5 above pH 7.5), and with free energies of heme-heme interaction (4.7 kJ.mole-1 at pH 7.2) which resemble those in other vertebrate hemoglobins that exhibit higher n50 values. Curiously, chloride ions increase hemoglobin-oxygen affinity below pH +/- 7.2. The possible mechanisms underlying the effects of ATP and chloride are discussed.

Adenosine Triphosphate↗
PubMed1989

Transtracheal Doppler: a new procedure for continuous cardiac output measurement.

An endotracheal tube with a 5 mm diameter ultrasonic transducer incorporated into its distal end was inserted into the tracheas of six anesthetized dogs. The device provides for ultrasound measurement of aortic diameter and blood velocity in the ascending aorta to obtain cardiac outputs. Because measurements are made proximal to the origins of the aortic arch vessels, total cardiac output is obtained. Procedures for measurement of aortic diameter, aortic blood velocity, and cardiac output are discussed. The resulting data were used to empirically determine the angle of incidence of the ultrasound beam that gave the best least squares fit over a range of thermodilution cardiac outputs. For an angle of incidence of 62.3 degrees, the relationship between cardiac output determined by thermal dilution and by transtracheal Doppler was transtracheal Doppler cardiac output = -.69 + 1.19 thermal dilution cardiac output. R2 = .82. The results from this preliminary assessment of the feasibility of measuring cardiac output in dogs using transtracheal Doppler suggest that additional studies should be pursued. Additional issues that require investigation include confirmation of constancy of human anatomy and demonstration of lack of tracheal trauma from the Doppler equipped endotracheal tube.

Animals↗
PubMed1988

Functional characterization of fetal and adult yak hemoglobins: an oxygen binding cascade and its molecular basis.

In contrast to most other mammals, the yak, which is native to high altitudes, has two major fetal and two or four major adult hemoglobin (Hb) components. We report the oxygen affinities and sensitivities to pH and 2,3-diphosphoglycerate of the two fetal and two adult Hbs commonly found in calves, compared to those of adult cow Hb A, and relate these findings to their primary structures and to placental maternal-fetal oxygen transfer at altitude. Arranged in order of decreasing oxygen affinity the Hbs are F1 (alpha I2 gamma 2), F2 (alpha II2 gamma 2), A1 (alpha II2 beta II2), and cow Hb A. The higher affinity of the fetal than the adult yak Hbs correlates with the beta 15Trp----Phe substitution, whereas the higher affinity in yak than in cow Hb correlates with the beta 135Ala----Val substitution. The difference in oxygen affinities between yak Hbs A1 and A2, which have identical beta chains, suggests the existence of yet unknown mechanisms determining oxygen affinity. The larger Bohr effects of F2 than F1 and of A2 than A1 are attributable to alpha-chain differences, most probably the alpha I50Glu----alpha II50His substitution.

Altitude↗
PubMed1988

High-altitude respiration of birds. Structural adaptations in the major and minor hemoglobin components of adult Rüppell's Griffon (Gyps rueppellii, Aegypiinae): a new molecular pattern for hypoxic tolerance.

The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted.

Acclimatization↗
PubMed1988

High altitude and hemoglobin function in the vultures Gyps rueppellii and Aegypius monachus.

Functional characteristics of the stripped composite hemoglobins (Hbs) of the vultures Gyps rueppellii and Aegypius monachus that can fly at extremely high altitudes, and of component Hbs of G. rueppellii are reported, in relation to influences of pH, temperature and inositol hexaphosphate. G. rueppellii Hbs A, A' and D represent a sequence of increasing oxygen affinity, which is opposite to earlier results on avian Hb components, but correlates with two alpha-chain substitutions that predictably affect oxygen affinity. The homo- and heterotropic interactions in oxygen binding are related to primary structures of the constituent polypeptide chains to trace molecular adaptations to high-altitude respiration, and to physiological factors (pulmonary hypoxia and hypocapnia, body temperature shifts, and lung and nasal gas and heat exchange) to discern their possible survival value at altitudes of 11,300 m.

Acclimatization↗
PubMed1987

Thermodynamic analysis of precisely measured oxygen equilibria of tench (Tinca tinca) hemoglobin and their dependence on ATP and protons.

Precise oxygen equilibria including extreme, high and low saturation values were determined for hemoglobin (Hb) from the freshwater teleost Tinca tinca at three temperatures, each at two pH levels and in the presence and absence of the erythrocytic cofactor ATP, at twofold molar excess over Hb. Analysis of the data in terms of Adair's successive oxygenation theory shows that in the absence of ATP, each of the four oxygenation steps are exothermic, but that net heat release decreases as pH falls from 8.2 to 7.4. ATP greatly depresses the temperature sensitivity of oxygenation particularly at physiological erythrocytic pH, where endothermic cofactor dissociation finds expression in a reverse temperature sensitivity for binding of the 3rd oxygen molecule to the tetrameric Hb. Enthalpy (delta Hi) and entropy (delta Si) changes of oxygenation vary with oxygenation step, i, as well as with pH and ATP addition, but the variations of delta Hi are similar to those of delta Si reflecting enthalpy-entropy compensation. The data show that the cooperative effects in tench Hb can be dominated either by entropic or enthalpic contributions, depending on the experimental condition and the oxygenation step.

Adenosine Triphosphate↗
PubMed1987

Analysis of teleost hemoglobin by Adair and Monod-Wyman-Changeux models. Effects of nucleoside triphosphates and pH on oxygenation of tench hemoglobin.

The allosteric effects of the erythrocytic nucleoside triphosphates (NTP) and of proton concentrations were investigated by precise measurement of Hb-O2 equilibria of tench hemoglobin (including extreme, high and low saturation ranges) and analysed in terms of the MWC two state model and the Adair four step oxygenation theory. At low concentrations (NTP/Hb ratio = 1.0, and pH greater than 7.3) ATP, GTP and protons decrease Hb-O2 affinity by increasing the allosteric constant L and reducing KT, the association constant of the deoxy, tense state of the Hb, without significantly affecting that (KR) of the oxy state, increasing the free energy of cooperativity (delta G). High concentrations of these effectors, however, also reduce KR. The greater sensitivity of the half-saturation O2 tension (P50) of the Hb to GTP than to ATP at the same concentration, correlates with greater effects of GTP on both KT and KR. The pH and NTP dependence of the four Adair association constants and the calculated fractional populations of Hb molecules in different stages of oxygenation show that the autochthonous NTP effectors and protons stabilize the T structure and postpone the T----R transition basic to cooperativity in fish Hb. The possible implications of the findings for aquatic respiration are discussed.

Animals↗