A spatial and temporal analysis of paediatric central nervous system infections from 2005 to 2015 in Ho Chi Minh City, Vietnam.

Central nervous system infections (CNSI) are a leading cause of death and long-term disability in children. Using ICD-10 data from 2005 to 2015 from three central hospitals in Ho Chi Minh City (HCMC), Vietnam, we exploited generalized additive mixed models (GAMM) to examine the spatial-temporal distribution and spatial and climatic risk factors of paediatric CNSI, excluding tuberculous meningitis, in this setting. From 2005 to 2015, there were 9469 cases of paediatric CNSI; 33% were ⩽1 year old at admission and were mainly diagnosed with presumed bacterial CNSI (BI) (79%), the remainder were >1 year old and mainly diagnosed with presumed non-bacterial CNSI (non-BI) (59%). The urban districts of HCMC in proximity to the hospitals as well as some outer districts had the highest incidences of BI and non-BI; BI incidence was higher in the dry season. Monthly BI incidence exhibited a significant decreasing trend over the study. Both BI and non-BI were significantly associated with lags in monthly average temperature, rainfall, and river water level. Our findings add new insights into this important group of infections in Vietnam, and highlight where resources for the prevention and control of paediatric CNSI should be allocated.

WAXS studies of the structural diversity of hemoglobin in solution.

Specific ligation states of hemoglobin are, when crystallized, capable of taking on multiple quaternary structures. The relationship between these structures, captured in crystal lattices, and hemoglobin structure in solution remains uncertain. Wide-angle X-ray solution scattering (WAXS) is a sensitive probe of protein structure in solution that can distinguish among similar structures and has the potential to contribute to these issues. We used WAXS to assess the relationships among the structures of human and bovine hemoglobins in different liganded forms in solution. WAXS data readily distinguished among the various forms of hemoglobins. WAXS patterns confirm some of the relationships among hemoglobin structures that have been defined through crystallography and NMR and extend others. For instance, methemoglobin A in solution is, as expected, nearly indistinguishable from HbCO A. Interestingly, for bovine hemoglobin, the differences between deoxy-Hb, methemoglobin and HbCO are smaller than the corresponding differences in human hemoglobin. WAXS data were also used to assess the spatial extent of structural fluctuations of various hemoglobins in solution. Dynamics has been implicated in allosteric control of hemoglobin, and increased dynamics has been associated with lowered oxygen affinity. Consistent with that notion, WAXS patterns indicate that deoxy-Hb A exhibits substantially larger structural fluctuations than HbCO A. Comparisons between the observed WAXS patterns and those predicted on the basis of atomic coordinate sets suggest that the structures of Hb in different liganded forms exhibit clear differences from known crystal structures.

Probing the importance of the amino-terminal sequence of the beta- and gamma-chains to the properties of normal adult and fetal hemoglobins.

A recombinant mutant of human fetal hemoglobin (Hb F), named rHb Oscar, has been constructed to explore the importance of the sequence of the amino-terminal region of the gamma-chain to the structural and functional properties of Hb F as compared to human normal adult hemoglobin (Hb A). Substitutions in the N-terminal region of Hb A have shown this region to be important to its structural and functional properties. Recent studies of recombinant mutants of Hb A with gamma-chain mutations have been used to probe the significance of the N-terminal sequence to the properties of Hb F. One of these mutants of Hb A, called rHb Felix, contains eight substitutions in the N-terminal region of the beta-chain corresponding to the sequence of the gamma-chain in that region [Dumoulin et al. (1998) J. Biol. Chem. 273, 35032-35038]. rHb Felix exhibits a 2,3-bisphosphoglycerate (2,3-BPG) response like that of Hb A, but its tetramer-dimer dissociation constant is similar to that of Hb F. In contrast, rHb Oscar contains a gamma-chain with eight mutations at the N-terminal end corresponding to the sequence of the beta-chain of Hb A in that region. (1)H NMR studies of rHb Oscar indicate a global structure like that of Hb F. rHb Oscar is not as stable against alkaline denaturation as Hb F but is more stable than Hb A, and it exhibits a stronger response to 2,3-BPG and inositol hexaphosphate as compared to Hb F. The 2,3-BPG effect in rHb Oscar also appears to be slightly enhanced compared to that in Hb A. Subzero isoelectric focusing experiments suggest that rHb Oscar does not have dissociation properties like those of Hb A. These results along with those of rHb Felix illustrate that the effects of the N-terminal region on structure and function of the Hb molecule are complicated by interactions with the rest of the molecule that are not yet well defined. However, studies of complementary mutations of Hb A and Hb F may eventually help to define such interactions and lead to a better understanding of the relationship between the amino acid sequence and the properties of the Hb molecule.

Distal ligand reactivity and quaternary structure studies of proximally detached hemoglobins.

The linkage between the proximal histidines and the proximal polypeptide in normal adult human hemoglobin (Hb A) has been proposed to play a major role in transmitting allosteric effects between oxygen binding sites [Perutz, M. F. (1970) Nature 228, 726-734]. Here we present circular dichroism (CD), (1)H NMR, analytical ultracentrifugation, and stopped-flow kinetic data to better define the quaternary structure of hemoglobins in which the linkage between the proximal histidines and the polypeptide backbone has been broken [Barrick et al. Nat. Struct. Biol. 4, 78-83 (1997)] and to characterize the distal ligand binding properties of these proximally detached Hbs. CD spectroscopy indicates that rHb (alphaH87G) and rHb (alphaH87G/betaH92G) retain at least partial T-quaternary structure with distal ligand bound, whereas rHb (betaH92G) does not, consistent with (1)H NMR spectra. Analytical ultracentrifugation reveals significant tetramer dissociation in rHb (betaH92G) to be the likely cause of loss of T-state markers. These quaternary structure studies indicate that in distally liganded Hb, the T-state is compatible with proximal linkages in the beta- but not the alpha-chains. (1)H NMR titrations of rHb (alphaH87G) with n-butyl isocyanide demonstrate the alpha-chains to be of high affinity as compared with the beta-chains. Comparing ligand association and dissociation rates between the rHb (alphaH87G) variant with the T- and R-states of wild-type Hb A indicates that at the alpha-chains, carbon monoxide affinity is modulated entirely by the proximal linkage, rather than from distal interactions. Some residual allosteric interactions may remain operative at the beta-chains of rHb (alphaH87G).

An additional H-bond in the alpha 1 beta 2 interface as the structural basis for the low oxygen affinity and high cooperativity of a novel recombinant hemoglobin (beta L105W).

Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.

Novel recombinant hemoglobin, rHb (beta N108Q), with low oxygen affinity, high cooperativity, and stability against autoxidation.

Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule. rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K). It has been reported by Olson and co-workers [Carver et al. (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin. Hence, we have further introduced this mutation, alpha L29F, into beta N108Q. rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A. The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F. (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature. On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system.

NMR reveals hydrogen bonds between oxygen and distal histidines in oxyhemoglobin.

Compared with free heme, the proteins hemoglobin (Hb) and myoglobin (Mb) exhibit greatly enhanced affinity for oxygen relative to carbon monoxide. This physiologically vital property has been attributed to either steric hindrance of CO or stabilization of O(2) binding by a hydrogen bond with the distal histidine. We report here the first direct evidence of such a hydrogen bond in both alpha- and beta-chains of oxyhemoglobin, as revealed by heteronuclear NMR spectra of chain-selectively labeled samples. Using these spectra, we have assigned the imidazole ring (1)H and (15)N chemical shifts of the proximal and distal histidines in both carbonmonoxy- and oxy-Hb. Because of their proximity to the heme, these chemical shifts are extremely sensitive to the heme pocket conformation. Comparison of the measured chemical shifts with values predicted from x-ray structures suggests differences between the solution and crystal structures of oxy-Hb. The chemical shift discrepancies could be accounted for by very small displacements of the proximal and distal histidines. This suggests that NMR could be used to obtain very high-resolution heme pocket structures of Hb, Mb, and other heme proteins.

Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.

Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of non-equivalent subunits face the problem of severe resonance overlap, which can prevent the unambiguous assignment of resonances, a necessary step in interpreting the spectra. We report the application of a chain-selective labeling technique, in which one type of subunit is labeled at a time, to carbonmonoxy-hemoglobin A (HbCO A). This labeling method can be used to extend previous resonance assignments of key amino acid residues, which are important to the physiological function of hemoglobin. Among these amino acid residues are the surface histidyls, which account for the majority of the Bohr effect. In the present work, we report the results of two-dimensional heteronuclear multiple quantum coherence (HMQC) experiments performed on recombinant (15)N-labeled HbCO A. In addition to the C2-proton (H epsilon(1)) chemical shifts, these spectra also reveal the corresponding C4-proton (H delta(2)) resonances, correlated with the N epsilon(2) and N delta(1) chemical shifts of all 13 surface histidines per alpha beta dimer. The HMQC spectrum also allows the assignment of the H delta(1), H epsilon(1), and N epsilon(1) resonances of all three tryptophan residues per alpha beta dimer in HbCO A. These results indicate that heteronuclear NMR, used with chain-selective isotopic labeling, can provide resonance assignments of key regions in large, multimeric proteins, suggesting an approach to elucidating the solution structure of hemoglobin, a protein with molecular weight 64.5 kDa.

Interspecies hybrid HbS: complete neutralization of Val6(beta)-dependent polymerization of human beta-chain by pig alpha-chains.

Interspecies hybrid HbS (alpha(2)(P)beta(2)(S)), has been assembled in vitro from pig alpha-globin and human beta(S)-chain. The alpha(2)(P)beta(2)(S) retains normal tetrameric structure (alpha(2)beta(2)) of human Hb and an O(2) affinity comparable to that of HbS in 50 mM Hepes buffer; but, its O(2) affinity is slightly higher than that of HbS in the presence of allosteric effectors (chloride, DPG and phosphate). The (1)H-NMR spectroscopy detected distinct differences between the heme environments and alpha(1)beta(1) interfaces of pig Hb and HbS, while their alpha(1)beta(2) interfaces appear very similar. The interspecies hybrid alpha(2)(H)beta(2)(P) resembles pig Hb; the pig beta-chain dictated the conformation of the heme environment of the human alpha-subunit, and to the alpha(1)beta(1) interfaces of the hybrid. In the alpha(2)(P)beta(2)(S) hybrid, beta(S)-chain dictated the conformation of human heme environment to the pig alpha-chain in the hybrid; but the conformation of alpha(1)beta(1) interface of this hybrid is close to, but not identical to that of HbS. On the other hand, the alpha(1)beta(2) interface conformation is identical to that of HbS. More important, the alpha(2)(P)beta(2)(S) does not polymerize when deoxygenated; pig alpha-chain completely neutralizes the beta(S)-chain dependent polymerization. The polymerization inhibitory propensity of pig alpha-chain is higher when it is present in the cis alpha(P)beta(S) dimer relative to that in a trans alpha(P)beta(A) dimer. The semisynthetically generated chimeric pig-human and human-pig alpha-chains by exchanging the alpha(1-30) segments of human and pig alpha-chains have established that the sequence differences of pig alpha(31-141) segment can also completely neutralize the polymerization. Comparison of the electrostatic potential energy landscape of the alpha-chain surfaces of HbS and alpha(2)(P)beta(2)(S) suggests that the differences in electrostatic potential energy surfaces on the alpha-chain of alpha(2)(P)beta(2)(S) relative to that in HbS, particularly the ones involving CD region, E-helix and EF-corner of pig alpha-chain are responsible for the polymerization neutralization activity. The pig and human-pig chimeric alpha-chains can serve as blueprints for the design of a new generation of variants of alpha-chain(s) suitable for the gene therapy of sickle cell disease.

Assessment of roles of surface histidyl residues in the molecular basis of the Bohr effect and of beta 143 histidine in the binding of 2,3-bisphosphoglycerate in human normal adult hemoglobin.

Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr effect at pH 7.4 and about 55% of the acid Bohr effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr effect of the hemoglobin molecule.

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.

A biochemical and biophysical characterization of recombinant mutants of fetal hemoglobin and their interaction with sickle cell hemoglobin.

Three recombinant mutants of human fetal hemoglobin (Hb F) have been constructed to determine what effects specific amino acid residues in the gamma chain have on the biophysical and biochemical properties of the native protein molecule. Target residues in these recombinant fetal hemoglobins were replaced with the corresponding amino acids in the beta chain of human normal adult hemoglobin (Hb A). The recombinant mutants of Hb F included rHb F (gamma 112Thr --> Cys), rHb F (gamma 130Trp --> Tyr), and rHb F (gamma 112Thr --> Cys/gamma 130Trp --> Tyr). Specifically, the importance of gamma 112Thr and gamma 130Trp to the stability of Hb F against alkaline denaturation and in the interaction with sickle cell hemoglobin (Hb S) was investigated. Contrary to expectations, these rHbs were found to be as stable against alkaline denaturation as Hb F, suggesting that the amino acid residues mentioned above are not responsible for the stability of Hb F against the alkaline denaturation as compared to that of Hb A. Sub-zero isoelectric focusing (IEF) was employed to investigate the extent of hybrid formation in equilibrium mixtures of Hb S with these hemoglobins and with several other hemoglobins in the carbon monoxy form. Equimolar mixtures of Hb A and Hb S and of Hb A(2) and Hb S indicate that 48-49% of the Hb exists as the hybrid tetramer, which is in agreement with the expected binomial distribution. Similar mixtures of Hb F and Hb S contain only 44% hybrid tetramer. The results for two of our recombinant mutants of Hb F were identical to the results for mixtures of Hb F and Hb S, while the other mutant, rHb F (gamma 130Trp --> Tyr), produced 42% hybrid tetramer. The sub-zero IEF technique discussed here is more convenient than room-temperature IEF techniques, which require Hb mixtures in the deoxy state. These recombinant mutants of Hb F were further characterized by equilibrium oxygen binding studies, which indicated no significant differences from Hb F. While these mutants of Hb F did not have tetramer-dimer dissociation properties significantly altered from those of Hb F, future mutants of Hb F may yet prove useful to the development of a gene therapy for the treatment of patients with sickle cell anemia.

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.

Another report of adverse reactions to immediate-release nifedipine.

Numerous case reports in the literature describe adverse drug events associated with immediate-release nifedipine (IRN). In addition, several publications alerted health care professionals regarding the agent. However, it is still administered, and adverse reactions are still reported. Our patient suffered IRN-induced hypotension, myocardial ischemia, and mental status changes. We recommend that the use of IRN in treatment of hypertension should be either totally prohibited or severely restricted. The product should not be readily available for indiscriminate administration. Also, continuing education and training are required to alert all health care professionals to the serious dangers associated with this drug.

Correct assembly of human normal adult hemoglobin when expressed in transgenic swine: chemical, conformational and functional equivalence with the human-derived protein.

Structural and functional investigations of recombinant human hemoglobin A (HbA) isolated from the erythrocytes of transgenic swine coexpressing human alpha- and beta-globins have been carried out to authenticate its correct expression, post-translational processing and assembly. The HbA expressed in transgenic swine (TgHbA) is indistinguishable from the human-derived HbA in terms of its isoelectric pH, mass and elution pattern on a Mono S column. The chemical identity of the alpha- and beta-globin chains of TgHbA with the corresponding chains from human-derived HbA has been established by tryptic peptide mapping and amino acid sequencing. The proton NMR spectra of TgHbA have demonstrated that the conformational aspects of the protein around the heme pocket are indistinguishable from those of the control sample of HbA. The equivalence of the hydrogen bond pattern of TgHbA (in particular the inter-subunit surfaces) with that of authentic HbA has also been established by NMR studies. Consistent with these structural and conformational analyses, the TgHbA also exhibits complete functional equivalence with the human-derived HbA with respect to oxygen affinity, cooperativity, Bohr effect and allostery. Hence the studies presented here demonstrate that the transgenic swine system correctly transcribes the alpha- and beta-globin transgenes, translates the respective alpha- and beta-globin mRNA to generate the corresponding globin chains, carries out the correct cotranslational processing of the translated globin chains, inserts the heme into the globin chains in the same orientation as in the human-derived HbA and assembles the alpha- and beta-subunits into a functionally cooperative tetramer that exhibits a response to allosteric effectors identical with that of human-derived HbA. Thus, in the transgenic swine system, in vitro chemical manipulation steps such as those needed in the Escherichia coli and the yeast systems, to convert the rHbA expressed in these systems into forms functionally identical with that of the human-derived protein, are not needed. An additional advantage of the transgenic swine system is the stability of the transgenes over many generations. Hence the transgenic swine could serve as an excellent system for the production of human HbA (or its variants) for structure-function studies and for therapeutic applications.

Novel water-mediated hydrogen bonds as the structural basis for the low oxygen affinity of the blood substitute candidate rHb(alpha 96Val-->Trp).

One of the most promising approaches for the development of a synthetic blood substitute has been the engineering of novel mutants of human hemoglobin (Hb) A which maintain cooperativity, but possess lowered oxygen affinity. We describe here two crystal structures of one such potential blood substitute, recombinant (r) Hb(alpha 96Val-->Trp), refined to 1.9 A resolution in an alpha-aquomet, beta-deoxy T-state, and to 2.5 A resolution in a carbonmonoxy R-state. On the basis of molecular dynamics simulations, a particular conformation had been predicted for the engineered Trp residue, and the lowered oxygen affinity had been attributed to a stabilization of the deoxy T-state interface by alpha 96Trp-beta 99Asp hydrogen bonds. Difference Fourier maps of the T-state structure clearly show that alpha 96Trp is in a conformation different from that predicted by the simulation, with its indole side chain directed away from the interface and into the central cavity. In this conformation, the indole nitrogen makes novel water-mediated hydrogen bonds across the T-state interface with beta 101Glu. We propose that these water-mediated hydrogen bonds are the structural basis for the lowered oxygen affinity of rHb(alpha 96Val-->Trp), and discuss the implications of these findings for future molecular dynamics studies and the design of Hb mutants.

Drug microencapsulation by PLA/PLGA coacervation in the light of thermodynamics. 1. Overview and theoretical considerations.

Phase separation of poly(lactide) (PLA) and poly(lactide-co-glycolide) (PLGA), often called "coacervation" in the pharmaceutical field, is one of the classical methods for peptide drug microencapsulation in biodegradable polyesters. Although numerous studies have used this technique, the underlying physicochemical mechanisms of polyester coacervation under conditions of microsphere production have not been well-described yet. Moreover, the quality of microencapsulation in terms of drug loading efficiency and residual organic solvents is often not entirely satisfactory and depends greatly on the specific drug and polymer used. The first part of this contribution reviews briefly the scientific and patent literature on PLA/PLGA coacervation. Then, the underlying physicochemical principles of polyester coacervation are discussed and relevant thermodynamic models presented. More specifically, attempts were made to clarify the necessary characteristics of polymers, solvents, and coacervating and hardening agents for successful phase separation and microsphere formation. These basic considerations may contribute to a better understanding of the boundary conditions crucial for efficient drug microencapsulation by polyester coacervation.

Contribution of surface histidyl residues in the alpha-chain to the Bohr effect of human normal adult hemoglobin: roles of global electrostatic effects.

We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(alpha20His-->Gln or alpha:H20Q); r Hb(alpha:H50Q); r Hb(alpha:H72Q); r Hb(alpha:H89Q); and r Hb(alpha:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the alpha-chain to the alkaline Bohr effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the alpha-chain, alpha20, alpha50, alpha72, alpha89, and alpha112, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance spectra of Hb A with those of the r Hbs. For the assignment of the C2 proton resonances of alpha20His and alpha112His, we have used combinations of mutations to compensate for the spectral perturbations resulting from the single mutations, which obscure the resonance assignment. On the basis of the new findings, in solvent containing 0.1 M chloride, the overall contributions from surface histidyl residues of both the alpha- and beta-chain and from other previously identified alkaline Bohr groups account for approximately 75% of the observed Bohr effect at pH 7.3 (the maximum Bohr effect under the prescribed solvent conditions). Our results show that some histidyl residues contribute to the Bohr effect and some oppose the net Bohr effect. In some cases, the addition of anions can diminish or reverse the contributions of specific histidyl residues to the overall Bohr effect. Thus, the Bohr effect, a heterotropic effect, depends on the intricate arrangement and interactions of all hydrogen and anion binding sites in the hemoglobin molecule. It is an excellent example of global electrostatic effects in proteins.

A test of the role of the proximal histidines in the Perutz model for cooperativity in haemoglobin.

Human haemoglobin has long been a paradigm for cooperative ligand binding and allostery. Through analysis of the crystal structures of deoxyhaemoglobin and liganded haemoglobin, Perutz proposed a model for cooperativity in which the bond between the proximal histidine and the protein couples haem rearrangements to protein structure rearrangements. Here we test this model by deleting the bonds between the histidine imidazole side chain and the polypeptide. This detachment method allows us to determine directly the contribution of proximal histidine coupling to cooperativity of distal ligand binding. Proximal detachment significantly increases ligand affinity, reduces cooperativity, and prevents quaternary structure switching, in accord with the Perutz model. Residual cooperativity indicates that additional haem communication pathways exist that do not involve the proximal histidine coupling mechanism.

Production of human normal adult and fetal hemoglobins in Escherichia coli.

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.