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.     

AUGUR: a program to predict, display and analyze the tertiary structure of B-DNA

A UGUR is a program to predict, display and analyze the three-dimensionalstructure of B-DNA. The user can choose one of six models topredict the helical parameters of a given sequence. These parametersare then used to generate the coordinates of the DNA model inthree-dimensional space (trajectory). The trajectory can bedisplayed and rotated on a graphics terminal The trajectoryand helical parameters can also be searched for bends and structuralhomologues. Received on August 17, 1987; accepted on December 31, 1987     

Algae flocculation in reservoir water

Removal of algae in the reservoir water was studied by electroflocculation using a bipolar cell with aluminum electrodes and flocculation by treatment with commercial alum. Comparison of both the methods is discussed.     

Efficiency of recycled nitrogen from residues of maize (Zea mays), soybean (Glycine max) and moong (Vigna radiata) on wheat (Triticum aestivum) grain yield

Summary A laboratory incubation experiment followed by a greenhouse experiment was made in a silty clay loam at Pantnagar, India, to recycle plant utilizable N from crop residues such as maize stubble, soybean hay andmoong straw. The beneficial effect of recycled N was tested by a wheat crop. Soybean hay yielded the most NO₃–N upon mineralization and also gave the highest wheat grain yield. Maize stubble mineralized the least NO₃–N and gave the lowest grain yield.Moong straw occupied an intermediate position. An intervening period of 30–45 days would be required for the residues in question to release plant utilizable NO₃–N in sufficient quantities. From a practical view point, soybean hay appears to be an ideal choice of a residue capable of providing sufficient supplemental N for a succeding wheat crop and can be easily fitted into the prevalent cropping sequence.     

Molecular dynamics of sickle and normal hemoglobins

Molecular dynamics (MD) simulations have been carried out for 62.5 ps on crystal structures of deoxy sickle cell hemoglobin (HbS) and normal deoxy hemoglobin (HbA) using the CHARMM MD algorithm, with a time step of 0.001 ps. In the trajectory analysis of the 12.5–62.5 (50 ps) simulation, oscillations of the radius of gyration and solvent-accessible surface area were calculated. HbS exhibited a general contraction during the simulation, while HbA exhibited a nearly constant size. The average deviations of simulated structures from the starting structures were found to be 1.8 Å for HbA and 2.3 Å for HbS. The average rms amplitudes of atomic motions (atomic flexibility) were about 0.7 Å for HbA and about 1.0 Å for HbS. The amplitudes of backbone motion correlate well with temperature factors derived from x-ray crystallography. A comparison of flexibility between the α- and β-chains in both HbA and HbS indicates that the β-chains generally exhibited greater flexibility than the α-chains, and that the HbS β-chains exhibit greater flexibility in the N-terminal and D- and F-helix regions than do those of HbA. The average amplitude of backbone torsional oscillations was about 9°, a value comparable with that of other simulations, with enhanced torsional oscillation occurring primarily at the ends of helices or in loop regions between helices. Comparison of atomic flexibility and torsional oscillation results suggests that the increased β-chain flexibility results from relatively concerted motions of secondary structure elements. The increased flexibility may play an important role in HbS polymerization. Time course analysis of conformational energy of association, hydrogen bonding and hydrophobic bonding (as calculated from solvent accessibility) shows that all three of these factors contribute to the stability of subunit association for both hemoglobins. © 1993 John Wiley & Sons, Inc.     

Evolution of arthropod hemocyanins and insect storage proteins (hexamerins)

Crustacean and cheliceratan hemocyanins (oxygen-transport proteins) and insect hexamerins (storage proteins) are homologous gene products, although the latter do not bind oxygen and do not possess the copper- binding histidines present in the hemocyanins. An alignment of 19 amino acid sequences of hemocyanin subunits and insect hexamerins was made, based on the conservation of elements of secondary structure observed in X-ray structures of two hemocyanin subunits. The alignment was analyzed using parsimony and neighbor-joining methods. Results provide strong indications for grouping together the sequences of the 2 crustacean hemocyanin subunits, the 5 cheliceratan hemocyanin subunits, and the 12 insect hexamerins. Within the insect clade, four methionine- rich proteins, four arylphorins, and two juvenile hormone-suppressible proteins from Lepidoptera, as well as two dipteran proteins, form four separate groups. In the absence of an outgroup sequence, it is not possible to present information about the ancestral state from which these proteins are derived. Although this family of proteins clearly consists of homologous gene products, there remain striking differences in gene organization and site of biosynthesis of the proteins within the cell. Because studies on 18S and 12S rRNA sequences indicate a rather close relationship between insects and crustaceans, we propose that hemocyanin is the ancestral arthropod protein and that insect hexamerins lost their copper-binding capability after divergence of the insects from the crustaceans.      

植物抗病基因工程进展(续)

植物抗病基因工程进展（续）洪剑明印莉萍邱泽生（首都师范大学生物系北京１０００３７）ＰＲＯＧＲＥＳＳＯＦＧＥＮＥＴＩＣＥＮＧＩＮＥＥＲＩＮＧＦＯＲＰＬＡＮＴＤＩＳＥＡＳＥＲＥＳＩＳＴＡＮＣＥＨＯＮＧＪｉａｎＭｉｎｇＹＩＮＬｉＰｉｎｇＱＩＵＺｅＳｈ...     

Population structure in the American oyster as inferred by nuclear gene genealogies

Multiple haplotypes from each of three nuclear loci were isolated and sequenced from geographic populations of the American oyster, Crassostrea virginica. In tests of alternative phylogeographic hypotheses for this species, nuclear gene genealogies constructed for these haplotypes were compared to one another, to a mitochondrial gene tree, and to patterns of allele frequency variation in nuclear restriction site polymorphisms (RFLPs) and allozymes. Oyster populations from the Atlantic versus the Gulf of Mexico are not reciprocally monophyletic in any of the nuclear gene trees, despite considerable genetic variation and despite large allele frequency differences previously reported in several other genetic assays. If these populations were separated vicariantly in the past, either insufficient time has elapsed for neutral lineage sorting to have achieved monophyly at most nuclear loci, or balancing selection may have inhibited lineage extinction, or secondary gene flow may have moved haplotypes between regions. These and other possibilities are examined in light of available genetic evidence, and it is concluded that no simple explanation can account for the great variety of population genetic patterns across loci displayed by American oysters. Regardless of the source of this heterogeneity, this study provides an empirical demonstration that different sequences of DNA within the same organismal pedigree can have quite different phylogeographic histories.      

In vivo evaluation of [11C]TMI,a COX-2 selective PET tracer,in baboons

Overexpression of Cyclooxygenase-2 (COX-2) enzyme is associated with the pathogenesis of inflammation, cancers, stroke, arthritis, and neurological disorders. Because of the involvement of COX-2 in these diseases, quantification of COX-2 expression using Positron Emission Tomography (PET) may be a biological marker for early diagnosis, monitoring of disease progression, and an indicator of effective treatment. At present there is no target-specific or validated PET tracer available for in vivo quantification of COX-2. The objective of this study is to evaluate [¹¹C]TMI, a selective COX-2 inhibitor (Ki?≤?1?nM) in nonhuman primates using PET imaging. PET imaging in baboons showed that [¹¹C]TMI penetrates the blood brain barrier (BBB) and accumulates in brain in a somewhat heterogeneous pattern. Metabolite analyses indicated that [¹¹C]TMI undergoes no significant metabolism of parent tracer in the plasma for baseline scans, however a relative faster metabolism was found for blocking scan. All the tested quantification approaches provide comparable tracer total distribution volume (V_T) estimates in the range of 3.2–7?(mL/cm³). We observed about 25% lower V_T values in blocking studies with meloxicam, a nonselective COX-2 inhibitor, compared to baseline [¹¹C]TMI binding. Our findings indicate that [¹¹C]TMI may be a suitable PET tracer for the quantification of COX-2 in vivo. Further experiments are needed to confirm the potential of this tracer in COX-2 overexpressing models for brain diseases.     

Influence of semisynthetic modification of the scaffold of a contact domain of HbS on polymerization: role of flexible surface topology in polymerization inhibition

A new variant of HbS, HbS-Einstein with a deletion of segment α_23–26 in the B-helix, has been assembled by semisynthetic approach. B-helix of the α chain of cis αβ-dimer of HbS plays dominant role in the quinary interactions of deoxy HbS dimer. This B-helix is the primary scaffold that provides the orientation for the side chains of contact residues of this intermolecular contact domain. The design of HbS-Einstein has been undertaken to map the influence of perturbation of molecular surface topology and the flexibility of surface residues in the polymerization. The internal deletion exerts a strong inhibitory influence on Val-6 (β)-dependent polymerization, comparable to single contact site mutations and not for complete neutralization of Val-6(β)-dependent polymerization. The scaffold modification in cis-dimer is inhibitory, and is without any effect when present on the trans dimer. The flexibility changes in the surface topology in the region of scaffold modification apparently counteracts the intrinsic polymerization potential of the molecule. The inhibition is close to that of Le Lamentin mutation [His-20 (α) → Gln] wherein a mutation engineered without much change in flexibility of the contact domain. Interestingly, the chimeric HbS with swine–human chimeric α chain with multiple non-conservative mutations completely inhibits the Val-6(β)-dependent polymerization. The deformabilities of surface topology of chimeric HbS are comparable to HbS in spite of the multiple contact site mutations in the α-chain. We conclude that the design of antisickling Hbs for gene therapy of sickle cell disease should involve multiple mutations of intermolecular contact sites.