Recipients of blood from a donor with multiple HLA antibodies: a lookback study of transfusion-related acute lung injury.

BACKGROUND: The effects of transfusion of HLA antibodies to patients with corresponding antigens are not well known. STUDY DESIGN AND METHODS: Records of patients who received blood from previous donations of a donor implicated in a case of transfusion-related acute lung injury (TRALI) were examined. The donor had multiple HLA antibodies reactive with 96 percent of HLA Class I antigens and 88 percent of HLA Class II antigens. RESULTS: Among 103 patients (40 with a pretransfusion white blood cell [WBC] count of >/=3.5 x 10(9)/L), 1 patient met criteria for TRALI and had clinical evidence for diffuse alveolar hemorrhage. Among the subset of 55 patients (17 with a pretransfusion WBC count of 3.5 x 10(9)/L) with known HLA types, none developed TRALI even though 54 (98%) had one to five corresponding HLA antigens. In a subgroup of patients four of 62 patients with chest radiographs, developed new or worse bilateral infiltrates with implicated but not control units (p = 0.0625). CONCLUSION: Transfusion of HLA antibodies from this donor to nonneutropenic patients did not cause TRALI, but there was a trend of an association with new or worse bilateral pulmonary infiltrates. Further research is needed to determine why transfusion of HLA antibodies to recipients with corresponding antigens causes TRALI in some cases and not in others.

The crystal structure of thymidylate synthase from Pneumocystis carinii reveals a fungal insert important for drug design.

Thymidylate synthase from Pneumocystis carinii (PcTS) is an especially important drug target, since P. carinii is a fungus that causes opportunistic pneumonia infections in immune-compromised patients and is among the leading causes of death of AIDS patients. Thymidylate synthase (TS) is the sole enzyme responsible for the de novo production of deoxythymidine monophosphate and hence is crucial for DNA replication in every organism. Inhibitors selective for P. carinii TS over human TS would be greatly beneficial in combating this disease. The crystal structure of TS from P. carinii bound to its substrate, dUMP, and a cofactor mimic, CB3717, was determined to 2.6 A resolution. A comparison with other species of TS shows that the volume of the closed PcTS active-site is 20 % larger than that of five other TS closed active-sites. A two-residue proline insert that is strictly conserved among all fungal species of TS, and a novel C-terminal closing interaction involving a P. carinii-specific tyrosine residue are primarily responsible for this increase in volume. The structure suggests several options for designing an inhibitor specific to PcTS and avoiding interactions with human TS. Taking advantage of the residue substitutions of P. carinii TS over human TS enables the design of a selective inhibitor. Additionally, the larger volume of the active-site of PcTS is an important advantage for designing de novo inhibitors that will exclude the human TS active-site through steric hindrance.

Adf-1 is a nonmodular transcription factor that contains a TAF-binding Myb-like motif.

Adf-1 is an essential Drosophila melanogaster sequence-specific transactivator that binds the promoters of a diverse group of genes. We have performed a comprehensive mapping of the functional domains of Adf-1 to study the role of transactivators in the process of gene activation. Using a series of clustered point mutations and small deletions we have identified regions of Adf-1 required for DNA binding, dimerization, and activation. In contrast to most enhancer-binding factors, the Adf-1 activation regions are nonmodular and depend on an intact protein, including the Adf-1 DNA-binding domain, for activity. Like many transcriptional activators, Adf-1 contains a TFIID-binding domain that can interact with specific TAF subunits. Although TAFs are required for Adf-1-directed activation, TAF binding is not sufficient, suggesting that Adf-1 may direct multiple essential steps during activation. Interestingly, both the TAF-binding domain and the DNA-binding domain contain sequences homologous to those of the Myb family of DNA-binding domains. Thus, Adf-1 has evolved an unusual structure containing two versions of the Myb motif, one that binds DNA and one that binds proteins.

Refined structures of substrate-bound and phosphate-bound thymidylate synthase from Lactobacillus casei.

Crystal structures of two crystal forms of the complex of Lactobacillus casei (TS) with its substrate dUMP have been solved and refined at 2.55 A resolution. The two crystal forms differ by approximately 5% in the c-axis length. The TS-dUMP complexes are symmetric dimers with dUMP bound equivalently in both active sites. dUMP is non-covalently bound in the same conformation as in ternary complexes of TS with dUMP and cofactor or cofactor analogs. The same hydrogen bonds are made between TS and substrate in the binary and ternary complexes. We have also determined the 2.36 A crystal structure of phosphate-bound L. casei TS. This structure has been refined to an R-factor of 19.3% with highly constrained geometry. Refinement has revealed the locations of all residues in the protein, including the disordered residues 90 to 119, which are part of an insert found only in the L. casei and Staphylococcus aureus transposon Tn4003 TS sequences. The 2.9 A multiple isomorphous replacement (MIR) structure of L. casei TS in a complex with its substrate dUMP has been refined to a crystallographic R-factor of 15.5%. Reducing agents were withheld from crystallization solutions during MIR structure determination to allow heavy-metal labeling of the cysteine residues. Therefore, the active-site cysteine residue in this structure is oxidized and the dUMP is found at half-occupancy in the active site. No significant conformational difference was found between the phosphate-bound and dUMP-bound structures. The TS-dUMP structures were better ordered than the phosphate-bound TS or the oxidized TS-dUMP, particularly Arg23, which is clearly hydrogen-bonded to the phosphate group of dUMP. A large and a small P6(1)22 crystal form are observed for both phosphate-bound and dUMP-bound L. casei TS. The small cell forms of the phosphate-bound and dUMP-bound enzyme are isomorphous, whereas the cell constants of the larger cell form change slightly when dUMP is bound (c = 240 A versus c = 243 A). For both liganded and unliganded enzyme, conversion from the small to the large crystal form sometimes occurs spontaneously, and the crystal packing changes at a single interface. Conversion may be the result of a small change in pH in the mother liquor surrounding the crystal. A single intermolecular contact between symmetry-related Asp287 residues is disrupted on going from the small to the large crystal form.

Structures of thymidylate synthase with a C-terminal deletion: role of the C-terminus in alignment of 2'-deoxyuridine 5'-monophosphate and 5,10-methylenetetrahydrofolate.

Thymidylate synthase undergoes a major conformational change upon ligand binding, where the carboxyl terminus displays the largest movement (approximately 4 A). This movement from an "open" unliganded state to the "closed" complexed conformation plays a crucial role in the correct orientation of substrates and in product formation. The mutant lacking the C-terminal valine (V316Am) of the enzyme is inactive. X-ray crystal structures of V316Am and its complexes with dUMP, FdUMP, and both FdUMP and CH2H4folate are described. The structures show that ligands are bound within the active site, but in different modes than those in analogous, wild-type thymidylate synthase structures. The 2.7-A binary complex structures of V316Am with FdUMP and dUMP show that the pyrimidine and ribose moieties of the nucleotides are pivoted approximately 20 degrees around the 3'-hydroxyl compared to dUMP in the wild-type enzyme. The 2.7-A crystal structure of V316Am complexed with cofactor, CH2H4folate, and the substrate analog, FdUMP, shows these ligands bound in an open conformation similar to that of the unliganded enzyme. In this ternary complex, the imidazolidine ring of the cofactor is open and has reacted with water to form 5-HOCH2H4folate. 5-HOCH2H4folate is structural evidence for the 5-iminium ion intermediate, which is the proposed reactive form of CH2H4folate. The altered ligand binding modes observed in the three V316Am complex structures open new venues for the design of novel TS inhibitors.

Structure-based discovery of inhibitors of thymidylate synthase.

A molecular docking computer program (DOCK) was used to screen the Fine Chemical Directory, a database of commercially available compounds, for molecules that are complementary to thymidylate synthase (TS), a chemotherapeutic target. Besides retrieving the substrate and several known inhibitors, DOCK proposed putative inhibitors previously unknown to bind to the enzyme. Three of these compounds inhibited Lactobacillus casei TS at submillimolar concentrations. One of these inhibitors, sulisobenzone, crystallized with TS in two configurations that differed from the DOCK-favored geometry: a counterion was bound in the substrate site, which resulted in a 6 to 9 angstrom displacement of the inhibitor. The structure of the complexes suggested another binding region in the active site that could be exploited. This region was probed with molecules sterically similar to sulisobenzone, which led to the identification of a family of phenolphthalein analogs that inhibit TS in the 1 to 30 micromolar range. These inhibitors do not resemble the substrates of the enzyme. A crystal structure of phenolphthalein with TS shows that it binds in the target site in a configuration that resembles the one suggested by DOCK.

Home health care: delineation of research priorities and formation of a national network group.

The purposes of this national study were (a) to determine priority research questions in home health care nursing and (b) to develop a home health research network group. Collaborative brainstorming was used initially by an interdisciplinary research team to generate a list of research questions. The major content areas covered in the questionnaire were questions relating to home health care as a delivery system, patient classification, quality assurance, nursing diagnosis, and nursing professionalism. A survey of 450 practicing home health care nurses was completed. The questions were rated by the respondents on a scale of high, medium, or low research priority. The response rate was 52%, with home health nurses responding from 30 states. The results clearly indicated the top 10 research priorities for home health care nursing research. The formation of the home health care research network groups are also discussed.

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase.

Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.

Structure, multiple site binding, and segmental accommodation in thymidylate synthase on binding dUMP and an anti-folate.

The structure of Escherichia coli thymidylate synthase (TS) complexed with the substrate dUMP and an analogue of the cofactor methylenetetrahydrofolate was solved by multiple isomorphous replacement and refined at 1.97-A resolution to a residual of 18% for all data (16% for data greater than 2 sigma) for a highly constrained structure. All residues in the structure are clearly resolved and give a very high confidence in total correctness of the structure. The ternary complex directly suggests how methylation of dUMP takes place. C-6 of dUMP is covalently bound to gamma S of Cys-198(146) during catalysis, and the reactants are surrounded by specific hydrogen bonds and hydrophobic interactions from conserved residues. Comparison with the independently solved structure of unliganded TS reveals a large conformation change in the enzyme, which closes down to sequester the reactants and several highly ordered water molecules within a cavernous active center, away from bulk solvent. A second binding site for the quinazoline ring of the cofactor analogue was discovered by withholding addition of reducing agent during crystal storage. The chemical change in the protein is slight, and from difference density maps modification of sulfhydryls is not directly responsible for blockade of the primary site. The site, only partially overlapping with the primary site, is also surrounded by conserved residues and thus may play a functional role. The ligand-induced conformational change is not a domain shift but involves the segmental accommodation of several helices, beta-strands, and loops that move as units against the beta-sheet interface between monomers.

Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases.

The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.

Long-range electrostatic interactions can influence the folding, stability, and cooperativity of dihydrofolate reductase.

To test the possibility that long-range interactions might influence the folding and stability of dihydrofolate reductase, a series of single and double mutations at positions 28 and 139 were constructed and their urea-induced unfolding reactions studied by absorbance and circular dichroism spectroscopy. The alpha carbons of the two side chains are separated by 15 A in the native conformation. The replacement of Leu 28 by Arg and of Glu 139 by Gln resulted in additive effects on both kinetic and equilibrium properties of the reversible unfolding transition; no evidence for interaction was obtained. In contrast, the Arg 28/Lys 139 double replacement changed the equilibrium folding model from two state to multistate and showed evidence for interaction in one of the two kinetic phases detected in both unfolding and refolding reactions. The results can be explained in terms of a long-range, repulsive electrostatic interaction between the cationic side chains at these two positions.

Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli.

The role of the secondary structure in the folding mechanism of dihydrofolate reductase from Escherichia coli was probed by studying the effects of amino acid replacements in two alpha helices and two strands of the central beta sheet on the folding and stability. The effects on stability could be qualitatively understood in terms of the X-ray structure for the wild-type protein by invoking electrostatic, hydrophobic, or hydrogen-bonding interactions. Kinetic studies focused on the two slow reactions that are thought to reflect the unfolding/refolding of two stable native conformers to/from their respective folding intermediates [Touchette, N. A., Perry, K. M., & Matthews, C. R. (1986) Biochemistry 25, 5445-5452]. Replacements at three different positions in helix alpha B selectively alter the relaxation time for unfolding while a single replacement in helix alpha C selectively alters the relaxation time for refolding. This behavior is characteristic of mutations that change the stability of the protein but do not affect the rate-limiting step. In striking contrast, replacements in strands beta F and beta G can affect both unfolding and refolding relaxation times. This behavior shows that these mutations alter the rate-limiting step in these native-to-intermediate folding reactions. It is proposed that the intermediates have an incorrectly formed beta sheet whose maturation to the structure found in the native conformation is one of the slow steps in folding.

Folding of dihydrofolate reductase from Escherichia coli.

The urea-induced equilibrium unfolding transition of dihydrofolate reductase from Escherichia coli was monitored by UV difference, circular dichroism (CD), and fluorescence spectroscopy. Each of these data sets were well described by a two-state unfolding model involving only native and unfolded forms. The free energy of folding in the absence of urea at pH 7.8, 15 degrees C is 6.13 +/- 0.36 kcal mol-1 by difference UV, 5.32 +/- 0.67 kcal mol-1 by CD, and 5.42 +/- 1.04 kcal mol-1 by fluorescence spectroscopy. The midpoints for the difference UV, CD, and fluorescence transitions are 3.12, 3.08, and 3.18 M urea, respectively. The near-coincidence of the unfolding transitions monitored by these three techniques also supports the assignment of a two-state model for the equilibrium results. Kinetic studies of the unfolding and refolding reactions show that the process is complex and therefore that additional species must be present. Unfolding jumps in the absence of potassium chloride revealed two slow phases which account for all of the amplitude predicted by equilibrium experiments. Unfolding in the presence of 400 mM KCl results in the selective loss of the slower phase, implying that there are two native forms present in equilibrium prior to unfolding. Five reactions were observed in refolding: two slow phases designated tau 1 and tau 2 that correspond to the slow phases in unfolding and three faster reactions designated tau 3, tau 4, and tau 5 that were followed by stopped-flow techniques. The kinetics of the recovery of the native form was monitored by following the binding of methotrexate, a tight-binding inhibitor of dihydrofolate reductase, at 380 nm.(ABSTRACT TRUNCATED AT 250 WORDS)