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C Poyart

Publications and source records attributed to C Poyart.

At least 37 records · Page 2Linked to original sources
PubMed1998

Chimeric beta-EF3-alpha hemoglobin (Psi): energetics of subunit interaction and ligand binding.

Among the numerous strategies to design an oxygen carrier, we outline in this work the engineering of a stable homotetrameric hemoglobin, expressed in Escherichia coli. The chimeric globin (Psi) consists of the first 79 residues of human beta globin (corresponding to positions NA1 --> EF3) followed by the final 67 residues of human alpha globin (corresponding to positions EF3 --> HC3). The molecular mass for beta-EF3-alpha (Psi) globin was measured using mass spectrometry to be equal to its theoretical value: 15782 Da. Correct protein folding was assessed by UV/visible and fluorescence spectra. The subunit interaction free energies were estimated by HPLC gel filtration. In the cyanometHb species, the formation of the dimer-tetramer interface is 2 kcal/mol less favorable (Delta G = -7 kcal/mol) than that of Hb A (Delta G = -9 kcal/mol), whereas the dimer-monomer interface is tightly assembled (< -10 kcal/mol) as for the Hb A alpha 1 beta 1 interface. In contrast to Hb A, oxygen binding to Psi Hb is not cooperative. The free energy for binding four oxygen molecules to a Psi homotetramer is slightly increased compared to a Hb A heterotetramer (-28 and -27.5 kcal/4 mol of O2, respectively). The intrinsic O2 affinity of a Psi homodimer is 6-fold higher than that of a homotetramer. The linkage scheme between dimer-tetramer subunit assembly and the noncooperative oxygenation of Psi Hb predicts a stabilization of the tetramer after ligand release. This protein mechanism resembles that of Hb A for which the dimers exhibit a 100-fold higher O2 affinity relative to deoxy tetramers (which are 10(5) times more stable than oxy tetramers). A potent allosteric effector of Hb A, RSR4, binds to Psi Hb tetramers, inducing a decrease of the overall O2 affinity. Since RSR4 interacts specifically with two binding sites of deoxy Hb A, we propose that the chimeric tetramer folding is close to this native structure.

Allosteric Regulation↗
PubMed1998

Fluorescent effector as a probe of the allosteric equilibrium in methemoglobin.

A fluorescent analogue of diphosphoglycerate (DPG), hydroxy-pyrenetrisulfonate (HPT), was used as a probe of the allosteric equilibrium of methemoglobin. Like DPG, HPT binds, one per tetramer, with a higher affinity to deoxyHb than to oxyHb. Once bound, the HPT fluorescence is quenched by energy transfer to the hemes. HPT can thus serve as a probe of the conformational state of the hemoglobin tetramer: a higher quenching indicates a stronger binding and therefore, more of the deoxy conformation. Since HPT binds to the same site as DPG, it can be displaced by DPG in order to determine the fluorescence intensity of the free HPT under the same conditions, to correct for the inner filter effect. The high spin ferric ligands, such as water and F, showed less fluorescence (more of the deoxy state) than low spin cyano-metHb. The aquo-metHb samples showed a reversion to the oxyHb conformation above pH 7, as expected due to the acid-alkaline transition forming hydroxy-metHb. Effectors such as bezafibrate, which do not bind to the same site as DPG, show an increase in the deoxy-like characteristics.

Allosteric Site↗
PubMed1998

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.

Adult↗
PubMed1998

Identifying the conformational state of bi-liganded haemoglobin.

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same alpha beta dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992). Science 255: 54-63]. The implications of this model and the relevant experiments will be reviewed here.

Allosteric Regulation↗
PubMed1998

Trematode hemoglobins show exceptionally high oxygen affinity.

Ligand binding studies were made with hemoglobin (Hb) isolated from trematode species Gastrothylax crumenifer (Gc), Paramphistomum epiclitum (Pe), Explanatum explanatum (Ee), parasitic worms of water buffalo Bubalus bubalis, and Isoparorchis hypselobagri (Ih) parasitic in the catfish Wallago attu. The kinetics of oxygen and carbon monoxide binding show very fast association rates. Whereas oxygen can be displaced on a millisecond time scale from human Hb at 25 degrees C, the dissociation of oxygen from trematode Hb may require a few seconds to over 20 s (for Hb Pe). Carbon monoxide dissociation is faster, however, than for other monomeric hemoglobins or myoglobins. Trematode hemoglobins also show a reduced rate of autoxidation; the oxy form is not readily oxidized by potassium ferricyanide, indicating that only the deoxy form reacts rapidly with this oxidizing agent. Unlike most vertebrate Hbs, the trematodes have a tyrosine residue at position E7 instead of the usual distal histidine. As for Hb Ascaris, which also displays a high oxygen affinity, the trematodes have a tyrosine in position B10; two H-bonds to the oxygen molecule are thought to be responsible for the very high oxygen affinity. The trematode hemoglobins display a combination of high association rates and very low dissociation rates, resulting in some of the highest oxygen affinities ever observed.

Animals↗
PubMed1998

Modulating the oxygen affinity of human fetal haemoglobin with synthetic allosteric modulators.

Improving the delivery of oxygen to the tissues by decreasing the oxygen affinity of haemoglobin has been a major aim of several laboratories over recent years because this may reduce the consequences of anaemia and/or improve tissue oxygenation in cases of decreased blood perfusion. Within the same context, lowering the oxygen affinity may prove valuable in the application of native or recombinant haemoglobin solutions as a blood substitute. The shift of the oxygen equilibrium curve to the right is obtained by various modulators. Among them, the bezafibrate derivatives are considered as a most interesting group. These principles are of the utmost importance in thalassaemia and other haemoglobinopathies where the beneficial effects of the compensatory synthesis of fetal haemoglobin are diminished by the increased oxygen affinity of this pigment. In this paper we present the results of a study initiated to determine whether a potent oxygen affinity modifier, RSR-4, could satisfactorily decrease the oxygen affinity of fetal haemoglobin, thus improving tissue oxygenation. The experiments were carried out on whole blood and on purified haemoglobin solutions and showed that the effector markedly decreased the oxygen affinity of HbF (from 18.7 to 3.73 mmHg in whole blood). At the same time the cooperativity index (n50) and the oxygen saturation levels remained within normal limits under the conditions of the main experiment. These observations have important implications for the potential application of oxygen affinity modifiers in vivo.

Aniline Compounds↗
PubMed1998

A new sickle cell disease phenotype associating Hb S trait, severe pyruvate kinase deficiency (PK Conakry), and an alpha2 globin gene variant (Hb Conakry).

A Guinean woman, heterozygous for haemoglobin (Hb) S, was studied because of episodes of marked anaemia, repeated typical metaphyseal painful crises and haemosiderosis. Her sickling syndrome resulted from the association of Hb S trait with a severe pyruvate kinase deficiency leading to a 2,3-DPG concentration of twice normal levels. Sequence of the PK-R gene revealed an undescribed mutation in the homozygous or hemizygous state within exon 5 (nucleotide 2670 C-->A), leading to the interchange of Ser 130 into Tyr (PK Conakry). In addition, the patient carried a new haemoglobin variant, Hb Conakry [alpha80(F1) Leu-->Val], which seemed to have a mild effect. The high intraerythrocytic 2,3-DPG concentration induced by the PK deficiency resulted in a decreased oxygen affinity which favoured sickling to a level almost similar to that of Hb S/C compound heterozygous patients. This was confirmed by oxygen binding measurements of Hb A/Hb S erythrocytes in which 2,3-DPG content was modified in vitro. Hysteresis between deoxy- and reoxygenation curves, as well as increase in the n(max) value, demonstrated that the extent of HbS polymerization in the propositus was almost the same as that of RBCs from a homozygous sickle cell patient or those of an A/S heterozygous patient with an artificial in vitro increase of 2,3-DPG concentration.

Adult↗
PubMed1998

A novel extended-spectrum TEM-type beta-lactamase (TEM-52) associated with decreased susceptibility to moxalactam in Klebsiella pneumoniae.

Klebsiella pneumoniae NEM865 was isolated from the culture of a stool sample from a patient previously treated with ceftazidime (CAZ). Analysis of this strain by the disk diffusion test revealed synergies between amoxicillin-clavulanate (AMX-CA) and CAZ, AMX-CA and cefotaxime (CTX), AMX-CA and aztreonam (ATM), and more surprisingly, AMX-CA and moxalactam (MOX). Clavulanic acid (CA) decreased the MICs of CAZ, CTX, and MOX, which suggested that NEM865 produced a novel extended-spectrum beta-lactamase. Genetic, restriction endonuclease, and Southern blot analyses revealed that the resistance phenotype was due to the presence in NEM865 of a 13.5-kb mobilizable plasmid, designated pNEC865, harboring a Tn3-like element. Sequence analysis revealed that the blaT gene of pNEC865 differed from blaTEM-1 by three mutations leading to the following amino acid substitutions: Glu104-->Lys, Met182-->Thr, and Gly238-->Ser (Ambler numbering). The association of these three mutations has thus far never been described, and the blaT gene carried by pNEC865 was therefore designated blaTEM-52. The enzymatic parameters of TEM-52 and TEM-3 were found to be very similar except for those for MOX, for which the affinity of TEM-52 (Ki, 0.16 microM) was 10-fold higher than that of TEM-3 (Ki, 1.9 microM). Allelic replacement analysis revealed that the combination of Lys104, Thr182, and Ser238 was responsible for the increase in the MICs of MOX for the TEM-52 producers.

Amino Acid Sequence↗
PubMed1998

Identification of streptococci to species level by sequencing the gene encoding the manganese-dependent superoxide dismutase.

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodA(int)) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus, S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus, S. canis, S. cricetus, S. downei, S. dysgalactiae, S. equi subsp. equi, S. equi subsp. zooepidemicus, S. equinus, S. gordonii, S. iniae, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis, S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis, S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodA(int) fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of their sodA(int) fragments on the phylogenetic tree of the sodA(int) fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

Base Sequence↗
PubMed1998

Synthesized allosteric effectors of the hemoglobin molecule: a possible mechanism for improved erythrocyte oxygen release capability in hemoglobinopathy H disease.

Patients with the nondeletion genotype of hemoglobinopathy H (HbH or beta4) disease have higher proportions of HbH and more severe tissue hypoxia than patients with the deletion genotype. Because these patients' red blood cells (RBCs) contain mainly two Hb species, HbH and HbA, the high proportion of HbA can be exploited by lowering its oxygen affinity; this would probably increase oxygen delivery to the RBCs and improve the patients' clinical phenotype. Allosteric effectors that induce a low-affinity Hb may be useful in this regard. We investigated the effect of a bezafibrate derivative, RSR-4, on the oxygen affinity of RBCs and purified hemolysates containing HbA and HbH. This allosteric effector crosses RBC membranes and binds reversibly to the alpha-chains of deoxy-Hb, decreasing hemoglobin oxygen affinity. The blood used was obtained from a patient with HbH disease (alphaTSaudi homozygote) whose HbH level was 33.5% as measured by high-performance liquid chromatography. Oxygen binding studies were performed in RBCs and purified hemolysates. RBCs incubated in the presence of 500 microM RSR-4 (2-[[[(3,5-dichloroanilino)-carbonyl]methyl]phenoxy]-2-methylpropi onic acid) in standard conditions (pH 7.4, 0.14 M NaCl, 37 degrees C) displayed an increase in their P50 value from 14.5 to 35.2 mm Hg. Oxygen binding studies in purified stripped hemolysates (pH 7.2, 0.1 M NaCl, 25 degrees C) showed that addition of both 500 microM RSR-4 and 1 mM of 2,3 diphosphoglycerate (DPG) led to an 11-fold decrease in oxygen affinity, whereas the addition of the natural effector DPG or RSR-4 alone produced a 2.7- and 5.7-fold decrease, respectively. In both cases, the oxygen equilibrium curves (OECs) were biphasic due to the presence of the noncooperative, high-oxygen-affinity HbH (beta4) component. After addition of RSR-4, the lower part of the OEC (corresponding to HbH) was not shifted compared with the upper part (corresponding to HbA). These results were confirmed by kinetic studies of CO recombination. Both experiments demonstrated that RSR-4 does not affect beta4 Hb. Our findings provide an experimental model for lowering the oxygen affinity of HbA in HbH-containing cells and suggest that the oxygen delivery capability of the latter would be thereby improved.

Allosteric Regulation↗
PubMed1997

Molecular characterization and expression analysis of the superoxide dismutase gene from Streptococcus agalactiae.

We have cloned and sequenced a 3103-bp DNA fragment carrying the gene encoding the Mn-SOD from Streptococcus agalactiae NEM318 serotype III. This DNA fragment contained four orfs that have the same polarity of transcription. Orf1 was truncated by molecular cloning and the corresponding 228-aa-long polypeptide did not exhibit any significant homology with other cognate proteins. Orf2 encodes a protein of 345 aa that displays some similarity (29% identity) with the YqeN peptide of Bacillus subtilis, the function of which is unknown. Orf3 encodes the 202-aa-long Mn-SOD which was functionally expressed in Escherichia coli. Orf4 was also truncated by molecular cloning and encodes 99 aa of the N-terminal moiety of a protein that displays significant homology (40% f identity) with the antiterminator LicT of B. subtilis. Transcriptional analysis revealed that the sodA gene of S. agalactiae NEM318 was transcribed monocistronically from a promoter, the activity of which is neither regulated by pH, O2, nor CO2 concentrations of the culture medium. Analysis by high resolution agarose gel electrophoresis of the AluI DNA polymorphism of the sodA locus in wild-type strains of S. agalactiae belonging to serogroups I, II, or III revealed no detectable difference.

Bacterial Proteins↗
PubMed1997

A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to beta-galactosidase in gram-positive bacteria.

A low-copy-number vector designated pTCV-lac has been constructed to provide a convenient system to analyze regulatory elements in Gram-positive bacteria. The main components of this vector are: (i) the origins of replication of pACYC184 and of the broad-host-range enterococcal plasmid pAM beta 1, (ii) erythromycin- and kanamycin-resistance-encoding genes for selection in Gram-negative and Gram-positive bacteria, (iii) the transfer origin of the IncP plasmid RK2, and (iv) a promoterless beta-galactosidase-encoding lacZ gene with a Gram-positive ribosome binding site. This 12 kb plasmid is present in Gram-positive hosts in three to five copies per chromosome equivalent and contains three unique cloning sites (EcoRI, SmaI, BamHI) for cloning of DNA inserts upstream of the lacZ gene. Plasmid pTCV-lac and derivatives carrying different promoter fragments have been transferred by conjugation from an Escherichia coli IncP mobilizing donor strain to Bacillus subtilis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus agalactiae. These plasmids were structurally stable in these hosts and the corresponding promoter activities, quantitated by the determination of the beta-galactosidase specific activities, were found to cover at least a 100-fold range in beta-galactosidase values. These results indicate that pTCV-lac should be useful for analysis of gene regulation in a wide range of Gram-positive bacteria.

Bacillus subtilis↗
PubMed1997

Influence of the A helix structure on the polymerization of hemoglobin S.

Hb S variants containing Lys-beta132 --> Ala or Asn substitutions were engineered to evaluate the consequences of the A helix destabilization in the polymerization process. Previous studies suggested that the loss of the Glu-beta7-Lys-beta132 salt bridge in the recombinant Hb betaE6V/E7A could be responsible for the destabilization of the A helix. The recombinant Hb (rHb) S/beta132 variants polymerized with an increased delay time as well as decreased maximum absorbance and Hb solubility values similar to that of Hb S. These data indicate that the strength of the donor-acceptor site interaction may be reduced due to an altered conformation of the A helix. The question arises whether this alteration leads to a true inhibition of the polymerization process or to qualitatively different polymers. The oxygen affinity of the beta132 mutated rHbs was similar to that of Hb A and S, whereas the cooperativity and effects of organic phosphates were reduced. This could be attributed to modifications in the central cavity due to loss of the positively charged lysine. Since Lys-beta132 is involved in the stabilization of the alpha1-beta1 interface, the loss of the beta132(H10)-beta128(H6) salt bridge may be responsible for the marked thermal instability of the beta132 mutated rHbs.

Biopolymers↗
PubMed1997

Recombinant [Phe(beta)63]hemoglobin shows rapid oxidation of the beta chains and low-affinity, non-cooperative oxygen binding to the alpha subunits.

We have engineered alpha2beta2 [Phe63]hemoglobin by changing the highly conserved distal histidine of the beta chains to a phenylalanine. The mutant tetramer binds four high-affinity ligands, such as CO or NO, to the ferrous form, or CN to the oxidized iron; however, it binds only two low-affinity ligands, oxygen and azide. The absorption spectrum of the ferrous deoxy or ferric forms are not normal, displaying an enhanced absorption of the visible band near 560 nm. Half of the autooxidation process, attributed to the mutated beta subunits, is over 1000-fold faster than for Hb A. The mutant Hb exhibits non-cooperative binding of two oxygens with an affinity about fivefold lower than those of HbA valency hybrids (alpha met beta)2. Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T., Hayashi, A., Shimizu, A. & Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280-282]. Flash-photolysis experiments also indicate non-cooperative behaviour: the CO-recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecular phase was the same for the (alpha(CO)metbeta)2 valency hybrid or the (alphaCO betaCO)2 form, suggesting mainly geminate CO-recombination kinetics to the beta chains. EPR and Resonance Raman spectra did not show evidence for a hemichrome, normally considered as a six-coordinated iron with low-spin character. The EPR and resonance Raman spectra for the mutated beta subunits demonstrate the presence of a high-spin compound in the ferric and deoxy ferrous forms. In particular, the ferrous mutated beta subunits are penta-coordinated. The abnormal absorption spectra are possibly due to an interaction between the porphyrin and the phenyl ring in the distal position rather than to direct binding to the iron.

Allosteric Regulation↗
PubMed1997

Alteration of an intersubunit contact in hemoglobin variants: comparative study of modifications at position alpha 126 Asp (H9).

Four human hemoglobin variants have already been described at position alpha 126 (H9), which is normally occupied by an aspartate: Hb Montefiore (-->Tyr), Hb Tarrant (-->Asn), Hb Fukutomi (-->Val), Hb Sassari (-->His). An additional variant, Hb West One (alpha 126 (H9) Asp-->Gly) is herein described. Aspartate alpha 126 (H9) is involved in a set of hydrogen bonds and salt bridges located at the C-terminal portion of the alpha-chains and of the C-helix of the beta-chains, which are broken in the oxy conformer, providing one of the most important sources of the difference in free energy between the T- and R-state in hemoglobin. A comparative study of four of these alpha 126 Hb variants is presented. An identical degree of alteration of the oxygen binding properties (increased oxygen affinity and decreased cooperativity) was found in all cases, when measured under standard experimental conditions (pH 7.2, 0.1 M NaCl). In contrast, the effect of L345 (a derivative of bezafibrate, which is a specific alpha-chain binding effector) on oxygen binding to Hb differed from one variant to another. When a bulky Tyr or His residue occupied the alpha 126 (H9) position, little effect of L345 was observed. Conversely, when this position was occupied by a residue of smaller size (Gly or Asn), normal heterotropic effects were observed. Molecular graphic modelling indicates that two classes of three-dimensional structure modifications may occur.

Allosteric Site↗
PubMed1997

Emergence of vancomycin resistance in the genus Streptococcus: characterization of a vanB transferable determinant in Streptococcus bovis.

Streptococcus bovis NEM760 was isolated from a stool swab collected on admission from a patient as surveillance for vancomycin-resistant enterococci. Strain NEM760 was identified as S. bovis by conventional biochemical methods and partial sequence analysis of its 16S rRNA. This strain was resistant to a low level of vancomycin (MIC, 64 micrograms/ml) but was susceptible to teicoplanin (MIC, 1 micrograms/ml), and vancomycin induced resistance to both glycopeptides. The presence of a vanB-related gene in NEM760 was demonstrated in a PCR assay which enabled specific amplification of a 635-hp internal segment of vanB. Sequence analysis of the corresponding PCR product revealed that it was highly homologous (96% identity) to the prototype vanB sequence of Enterococcus faecalis V583. The VanB resistance of determinant of S. bovis NEM760 was transferred by conjugation to E. faecalis and Enterococcus faecium at a similar frequency of 2 x 10(-5) per donor. SmaI-digested genomic DNAs of independently obtained transconjugants of E. faecalis and E. faecium were analyzed by pulsed-field gel electrophoresis and Southern hybridization with a vanB DNA probe. The electrophoretic and hybridization patterns obtained with all transconjugants of the same species were indistinguishable and revealed vanB-containing chromosomal insertions of approximately 100 kb. These results suggest that the genes mediating VanB-type resistance in S. bovis NEM760 are part of large transferable genetic elements. The results presented in the report demonstrate for the first time the role of streptococci in the dissemination of vancomycin resistance among gram-positive bacteria.

Anti-Bacterial Agents↗
PubMed1996

Asymmetric hemoglobin hybrids.

We have investigated the functional properties of hemoglobin (Hb) valency hybrids, and specifically whether it makes a difference if the oxidized subunits are on the same dimer (asymmetric hybrid) or not. CO recombination kinetics were used to probe the allosteric equilibrium of tetramers with two oxidized subunits. Asymmetric hybrids were prepared by mixing HbCO with a large excess of cyano-metHb; dimer exchange occurs in the order of seconds, producing a population of fully liganded [dimer-CO/dimer-CN] hybrids; these hybrids could then be photolysed to study CO rebinding to the doubly liganded [dimer-deoxy/dimer-CN] species. Before mixing, the HbCO samples typically show 30 to 50% slow phase, characteristic of deoxy (or T-state) Hb. Addition of HbCN to these samples decreased the slow fraction. The higher the ratio of HbCN to HbCO, the less slow phase was observed, with about 5% slow phase at a ratio of 10:1. This would indicate that the photoproduct [deoxy-dimer/ dimer-CN] is not predominantly in the low-affinity (T-state) conformation. We did not observe the difference between asymmetric and symmetric hybrids expected from published studies. The difference between the present flash photolysis results and the published equilibrium studies could be due to a kinetic factor: if the conversion to the T-state is slow after photolysis, then the biomolecular kinetics will reflect the pre-flash conditions of fully liganded (R-state) hemoglobin.

Allosteric Regulation↗