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Biomedical subjects

B Bohn

Publications and source records attributed to B Bohn.

At least 19 recordsLinked to original sources
PubMed1999

[Evaluation of esophageal motility disorders triggered by ingestion of solids in the case of non-obstructive dysphagia].

UNLABELLED: The aim of this study was to define the normal manometric pattern of esophageal motility in response to food ingestion and to evaluate the contribution of esophageal manometry in the management of patients complaining of functional dysphagia. PATIENTS AND METHODS: Twenty-one healthy volunteers and 25 consecutive patients complaining of functional dysphagia with normal conventional esophageal manometry were included in this prospective study. An event marker was used to study the relationship between dysphagia and motility events. RESULTS: Twenty-two out of 25 patients (88%) reported dysphagia during esophageal manometry with food ingestion, while none complained of dysphagia during conventional esophageal manometry. Significantly, food ingestion induced in healthy volunteers and in patients: an increase in the amplitude and duration of esophageal body peristaltic contractions, and a decrease in their propagation speed; an increase in the basal pressure and a decrease in the relaxation percentage of the lower esophageal sphincter during deglutition. The percentage of solid swallows with one or several of the 7 abnormal motility patterns studied prospectively was significantly higher among patients (53.7%) than among healthy volunteers (4.3%) (P < 0.0001); it was also significantly higher among patients during swallows with dysphagia (70.1%) than without dysphagia (33.6%) (P < 0.0001). CONCLUSION: Esophageal manometry with food ingestion is an effective means of defining abnormal motility patterns and their relationship with dysphagia during functional dysphagia.

Adult↗
PubMed1998

Characteristics of patients with dual infection by hepatitis B and C viruses.

BACKGROUND/AIMS: The purpose of this study was to compare the epidemiological, biochemical, virological and histological characteristics of patients with chronic hepatitis B and C with those of patients suffering from chronic hepatitis C alone. METHODS: Twenty-three patients with chronic hepatitis C, who were anti-HCV positive and HBs antigen positive, were studied and subdivided into two groups according to the presence or absence of HBV DNA replication. They were compared to 69 age- and sex-matched patients with chronic hepatitis who were anti-HCV positive and HBs antigen negative. All patients were HCV RNA positive by PCR, anti-HIV negative and anti-HDV negative. HBV DNA and HCV RNA were detected in serum by means of a branched DNA assay and PCR. The HCV serotypes were determined by the Chiron Riba HCV serotyping SIA technique. The histological characteristics included the Knodell score. RESULTS: Epidemiological, biochemical and virological parameters were not different between the two groups. Only the prevalence of cirrhosis was greater in chronic hepatitis B and C patients than in patients with chronic hepatitis C alone (p = 0.01). Among chronic hepatitis B and C patients, HCV RNA level was significantly lower in HBV DNA positive than in HBV DNA negative patients (p = 0.01). Indeed, histological lesions were more severe in HBV DNA positive than in HBV DNA negative patients, including prevalence of cirrhosis (p = 0.01), Knodell score (p = 0.05) and, among the latter, piecemeal necrosis (p = 0.01) and fibrosis (p = 0.05). The characteristics of patients with dual infection did not differ according to the mode of contamination and duration of HBV disease, except for a shorter duration in patients contaminated by drug abuse than in other patients. CONCLUSIONS: These results suggest that HBV DNA replication inhibits HCV RNA replication in patients with chronic active hepatitis B and C but increases the severity of histological lesions.

Adult↗
PubMed1995

Evidence for sub-picosecond heme doming in hemoglobin and myoglobin: a time-resolved resonance Raman comparison of carbonmonoxy and deoxy species.

Separation of the photophysical aspects of the sub-picosecond (sub-ps) time-resolved resonance Raman signal from contributions due to conformation has been achieved by comparing deoxyhemoglobin (Hb) in the T state with (carbonmonoxy)hemoglobin (HbCO), deoxy-beta 4 (beta 4 CO) (All R state), and monomers deoxymyoglobin and (carbonmonoxy)myoglobin (MbCO) [beta 4 consists of a tetramer of four beta-subunits and shows no cooperativity]. In all photolyzed species, Hb*(CO), Mb*(CO), and beta 4*(CO), the iron-histidine out-of-plane mode (vFe-His), indicative of heme doming, achieves 90% of its full intensity in 1 ps. The frequency of this mode (223-228 cm-1) is shifted significantly relative to equilibrium deoxy-Hb (210-216 cm-1) in the T state, but not with respect to either equilibrium deoxy-Mb or deoxy-beta 4. A correlation between the +12 cm-1 bandshift of vFe-His and the -2 cm-1 shift of the electron density marker band (v4 at 1370 cm-1) relative to T-state deoxy-Hb is shown to hold on all time scales, including the sub-picosecond time scale. Photolyzed Hb*(CO) consists of R-state or weakly interacting tetramers on the picosecond time scale and is shown to have properties similar to those of photolyzed Mb*(CO) and beta 4*(CO) on the picosecond time scale. These results establish that heme doming occurs as an ultrafast reaction to ligand dissociation and that heme doming is the primary event in the sequence of conformational changes leading to the cooperative R-->T transition.

Carboxyhemoglobin↗
PubMed1995

Functional aspects of ultra-rapid heme doming in hemoglobin, myoglobin, and the myoglobin mutant H93G.

Heme iron out-of-plane displacement following ligand dissociation in hemoglobin, myoglobin, and the proximal cavity mutant H93G is shown to be as rapid as the heme iron out-of-plane vibrational period by sub-picosecond time-resolved resonance Raman spectroscopy. The results demonstrate that the effect of steric repulsion initiated by the spin change of the iron gives rise to heme doming independent of covalent attachment of the proximal ligand to the protein. It is concluded that the protein plays a passive role in the initial ultrafast heme iron motion toward the out-of-plane position observed in the deoxy structure of hemoglobin and myoglobin. The results suggest that the spin change of the heme iron is the primary cause of rapid heme doming and that steric repulsion of the proximal ligand with the heme plays a secondary role in forcing the iron out of the heme plane.

Animals↗
PubMed1995

Ciprofloxacin and long-term prevention of spontaneous bacterial peritonitis: results of a prospective controlled trial.

The aim of this prospective double-blind study was to evaluate the value of long-term antibiotic prophylaxis using ciprofloxacin for the prevention of spontaneous bacterial peritonitis (SBP) in 60 cirrhotic patients with low ascitic fluid protein levels (< 15 g/L). The patients were assigned to two groups: group I (n = 28) ciprofloxacin 750 mg per os once a week for 6 months, group II (n = 32) placebo. The two groups were similar for clinical and laboratory characteristics. Twelve patients developed an intercurrent disorder, and 10 patients died during the trial. There were no adverse effects in the treated group. There was a significant decrease in the incidence of SBP (3.6 vs. 22%) (P < .05) and duration of hospitalization (9.3 +/- 4.5 vs. 17.6 +/- 6.2 days) (P < .05) in the treated group as compared with the placebo group. The bacteriological study showed no acquired resistance to ciprofloxacin after 6 months' treatment. These results suggest that long-term preventive antibiotic prophylaxis based on the weekly administration of 750 mg of ciprofloxacin is effective in the prevention of SBP in cirrhotic patients.

Adult↗
PubMed1994

Direct evidence for the role of haem doming as the primary event in the cooperative transition of haemoglobin.

The study of cooperative ligand binding among the four subunits of haemoglobin has played a central role in the understanding of allosteric transitions in a large number of enzymes. Haem iron out-of-plane motion has been suggested to be the trigger for the cooperative transition of haemoglobin. To function as a trigger in a dynamic sense, haem-iron doming must be the first conformational change to occur following ligand dissociation. Here we present the first direct demonstration that haem-iron doming occurs on the same time scale as the breaking of the iron-ligand bond, thus establishing haem-iron doming as the primary event which lead to the R-->T transition in haemoglobin.

Allosteric Regulation↗
PubMed1992

Allosteric modifiers of hemoglobin: 2-[4-[[(3,5-disubstituted anilino)carbonyl]methyl]phenoxy]-2-methylpropionic acid derivatives that lower the oxygen affinity of hemoglobin in red cell suspensions, in whole blood, and in vivo in rats.

Two new potent allosteric effectors of hemoglobin, RSR-4 [2-[4-[[(3,5-dichloroanilino)carbonyl]-methyl]phenoxy]-2- methylpropionic acid] and RSR-13 [2-[4-[[(3,5-dimethlanilino)carbonyl]methyl]-phenoxy]-2-methylp rop ionic, are compared to the previously reported compounds L3,5 and L3,4,5 [Lalezari, I., Lalezari, P., Poyart, C., Marden, M., Kister, J., Bohn, B., Fermi, G., & Perutz, M. F. (1990) Biochemistry 29, 1515]. Unlike L3,5 and L3,4,5, RSR-4 and RSR-13 are less impeded by physiological concentrations of serum albumin. RSR-4 has also been shown to be more effective than L3,5 in shifting the allosteric equilibrium of bovine Hb toward the low-affinity T-state. X-ray crystal studies show that both RSR-4 and RSR-13 bind to only one pair of symmetry-related sites in the Hb central water cavity whereas previous studies on L3,5 and L3,4,5 demonstrated a second pair of symmetry-related binding sites near Arg 104 beta. Three major interactions between these allosteric effectors and Hb include the acid group with the guanidinium group of C-terminal Arg 141 alpha, the effector's amide oxygen with the ammonium ion of Lys 99 alpha, and the phi electrons of the halogenated or methylated aromatic ring and Asn 108 beta. No explanation has been found for the difference in number of binding sites observed for RSR-4 and RSR-13 (two sites) compared to L3,5 and L3,4,5 (four sites); also no correlation has been made between the number of binding sites and degree of allosteric shift in the oxygen equilibrium curve.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Steric and hydrophobic determinants of the solubilities of recombinant sickle cell hemoglobins.

Models for the structure of the fibers of deoxy sickle cell hemoglobin (Hb Hb S, beta 6 Glu-->Val) have been obtained from X-ray and electron microscopic studies. Recent molecular dynamics calculations of polymer formation give new insights on the various specific interactions between monomers. Site-directed mutagenesis with expression of the Hb S beta subunits in Escherichia coli provides the experimental tools to test these models. For Hb S, the beta 6 Val residue is intimately involved in a specific lateral contact, at the donor site, that interacts with the acceptor site of an adjacent molecule composed predominantly of the hydrophobic residues Phe 85 and Leu 88. Comparing natural and artificial mutants indicates that the solubility of deoxyHb decreases in relation to the surface hydrophobicity of the residue at the beta 6 position with Ile > Val > Ala. We also tested the role of the stereospecific adjustment between the donor and acceptor sites by substituting Trp for Glu at the beta 6 location. Among these hydrophobic substitutions and under our experimental conditions, only Val and Ile were observed to induce polymer formation. The interactions for the Ala mutant are too weak whereas a Trp residue inhibits aggregation through steric hindrance at the acceptor site of the lateral contact. Increasing the hydrophobicity at the axial contact between tetramers of the same strand also contributes to the stability of the double strand. This is demonstrated by associating the beta 23 Val-->Ile mutation at the axial contact with either the beta 6 Glu-->Val or beta 6 Glu-->Ile substitution in the same beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1991

Allosteric transition in triply met-haemoglobin.

Methaemoglobin undergoes a transition to a T-like form at acid pH in the presence of strong effectors such as inositol hexakisphosphate (IHP), as evidenced by spectroscopic and oxidation potential measurements. Since oxygen and CO do not bind to the ferric haems, it is difficult to compare the properties of the R-met and T-met forms with those of ferrous haemoglobin. We have therefore prepared 90% oxidized samples, where the dominant signal for ligand (oxygen or CO) binding is due to tetramers with three met haems. Measurements were made of the oxygen equilibrium curves and CO rebinding kinetics after photodissociation. Without effectors, the partially oxidized samples show mainly R-state properties. Addition of IHP at acid pH induces an increase in T-state behaviour, as indicated by a lower oxygen affinity and a higher fraction of the slow bimolecular component for CO rebinding.

Allosteric Regulation↗
PubMed1991

Coupling of ferric iron spin and allosteric equilibrium in hemoglobin.

The allosteric transition in triply ferric hemoglobin has been studied with different ferric ligands. This valency hybrid permits observation of oxygen or CO binding properties to the single ferrous subunit, whereas the liganded state of the other three ferric subunits can be varied. The ferric hemoglobin (Hb) tetramer in the absence of effectors is generally in the high oxygen affinity (R) state; addition of inositol hexaphosphate induces a transition towards the deoxy (T) conformation. The fraction of T-state formed depends on the ferric ligand and is correlated with the spin state of the ferric iron complexes. High-spin ferric ligands such as water or fluoride show the most T-state, whereas low-spin ligands such as cyanide show the least. The oxygen equilibrium data and kinetics of CO recombination indicate that the allosteric equilibrium can be treated in a fashion analogous to the two-state model. The binding of a low-spin ferric ligand induces a change in the allosteric equilibrium towards the R-state by about a factor of 150 (at pH 6.5), similar to that of the ferrous ligands oxygen or CO; however, each high-spin ferric ligand induces a T to R shift by a factor of 40.

Allosteric Regulation↗
PubMed1990

Hemoglobin Calais [beta 76 (E20) Ala----Pro]: a hemoglobin variant with decreased intrinsic oxygen affinity.

Hb Calais [beta 76 (E20) Ala----Pro] is a new human hemoglobin variant displaying a decreased oxygen affinity. The only electrophoretical difference with Hb A was a slightly more acidic isoelectric point. A 2-fold decrease in the oxygen affinity was found by equilibrium measurements performed in a suspension of intact red blood cells and in the lysate. It was confirmed by kinetic studies of the purified abnormal hemoglobin. The rate of methemoglobin formation at 37 degrees C of Hb Calais was also increased relative to Hb A. The mechanism by which the Pro for Ala substitution of an external residue in the beta-chains results in these profound functional abnormalities is unclear. Subtle changes at the heme pocket, at a distance from the mutation, may be a plausible explanation for the effects observed.

Adult↗
PubMed1990

New effectors of human hemoglobin: structure and function.

We describe the actions of two new allosteric effectors of hemoglobin, 2-[4-(3,5-dichlorophenylureido)phenoxy]-2-methylpropionic acid (L35) and 2-[4-(3,4,5-trichlorophenylureido)phenoxy]-2-methylpropionic acid (L345). Each of them binds to two pairs of symmetry-related sites in the central cavity of human deoxyhemoglobin. One pair of sites overlaps with that occupied by bezafibrate [Perutz et al. (1986) J. Am. Chem. Soc. 108, 1064-1078]. The other sites are new, and the pair occupied by L35 is different from that occupied by L345. All the sites are at least 20 A from the site where organic phosphates are bound. L345 is by far the most potent allosteric effector of hemoglobin ever described. At a concentration of 0.1 mM, it raises the P50 of a suspension of red cells by 50%; at 0.2 mM it raises the P50 2.5-fold. At acid pH, it reduces Hill's coefficient to near unity and prevents complete oxygen saturation even under 1 atm of pure oxygen. In azidemethemoglobin at pH 6, it induces a transition to higher spin. These properties are reminiscent of those of teleost fish hemoglobins that exhibit a Root effect. The influence of L35 and L345 and that of organic phosphates on the oxygen affinity are additive, but they compete with chloride. L35 acts more weakly than L345, but can be made to induce the same effects as L345 alone by adding inositol hexaphosphate. Both compounds increase the alkaline and acid Bohr effects. They alter the bimolecular kinetics of CO recombination after a flash by increasing the slowly reacting fraction of hemoglobin in the T state at the expense of the fast-reacting fraction in the R state.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1990

Effectors of hemoglobin. Separation of allosteric and affinity factors.

The relative contributions of the allosteric and affinity factors toward the change in p50 have been calculated for a series of effectors of hemoglobin (Hb). Shifts in the ligand affinity of deoxy Hb and the values for 50% ligand saturation (p50) were obtained from oxygen equilibrium data. Because the high-affinity parameters (liganded conformation) are poorly determined from the equilibrium curves, they were determined from kinetic measurements of the association and dissociation rates with CO as ligand. The CO on-rates were obtained by flash photolysis measurements. The off-rates were determined from the rate of oxidation of HbCO by ferricyanide, or by replacement of CO with NO. The partition function of fully liganded hemoglobin for oxygen and CO is only slightly changed by the effectors. Measurements were made in the presence of the effectors 2,3-diphosphoglycerate (DPG), inositol hexakisphosphate (IHP), bezafibrate (Bzf), and two recently synthesized derivatives of Bzf (LR16 and L35). Values of p50 change by over a factor of 60; the on-rates decrease by nearly a factor of 8, with little change in the off-rates for the liganded conformation. The data indicate that both allosteric and affinity parameters are changed by the effectors; the changes in ligand affinity represent the larger contribution toward shifts in p50.

Allosteric Site↗
PubMed1990

Enhanced polymerization of recombinant human deoxyhemoglobin beta 6 Glu----Ile.

Polymerization of the deoxy form of sickle cell hemoglobin (Hb S; beta 6 Glu----Val) involves both hydrophobic and electrostatic intermolecular contacts. These interactions drive the mutated molecules into long fibrous rods composed of seven pairs of strands. X-ray crystallography of Hb S and electron microscopy image reconstruction of the fibers have revealed the remarkable complementarity between one of the beta 6 valines of each molecule (the donor site) and an acceptor site at the EF corner of a neighboring tetramer. This interaction constitutes the major lateral contact between the two strands in a pair. To estimate the relative importance of this key hydrophobic contact in polymer formation we have generated a polymerizing Hb with isoleucine at the beta 6 position (beta E6I) by site-directed mutagenesis. The mutated beta chains were produced in Escherichia coli and reassembled into functional tetramers with native alpha chains. Compared to native Hb S, the beta E6I mutant polymerizes faster and with a shortened delay time in 1.8 M phosphate buffer, indicating an increased stability of the nuclei preceding fiber growth. The solubility of the beta E6I mutant Hb is half that of native Hb S. Computer modeling of the donor-acceptor interaction shows that the presence of an isoleucine side chain at the donor site induces increased contacts with the receptor site and an increased buried surface area, in agreement with the higher hydrophobicity of the isoleucine residue. The agreement between the predicted and experimental differences in solubility suggests that the transfer of the beta 6 valine or isoleucine side chain from water to a hydrophobic environment is sufficient to explain the observations.

Base Sequence↗
PubMed1990

Haemoglobin alpha 2 beta 2 23Val----Ile produced in Escherichia coli facilitates Hb S polymerization.

The doubly substituted variant Hb S-Antilles (beta 6 Glu----Val, beta 23 Val----Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S-Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S-Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb alpha 2 beta 2 23 Val----Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1.8 M phosphate buffer. Mixtures of equal amounts of Hb S + Hb beta 23 Val----Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The beta 23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.

Escherichia coli↗
PubMed1989

Effect of ligand-affinity differences of human hemoglobin variants on electrophoretic behavior and their isolation and functional characterization.

A natural sulfated polysaccharide (agaropectin), contained in crude agar, can be used as a medium for electrophoretic separation of hemoglobin mutants, constituting a particular class of protein-ligand interactions. Mutations which either modify the electrostatic charge at the surface of the hemoglobin molecule or not, have been studied according to their putative interaction with the medium. Using conformational specificities of the hemoglobin molecule, we have also demonstrated that isoelectric focusing on a polyacrylamide gel in the absence of heme ligands represents a useful, convenient and rapid procedure for isolating silent Hb variants in their native form, provided that they exhibit an abnormal Bohr effect.

Calcium↗