Fatal pulmonary fibrosis associated with BCNU: the relative role of platelet-derived growth factor-B, insulin-like growth factor I, transforming growth factor-beta1 and cyclooxygenase-2.

Pulmonary fibrosis is a severe complication associated with bis-chloronitrosourea (BCNU) therapy. However, the pathogenetic mechanism has never been well investigated. We report here a 26-year-old female with diffuse large B-cell lymphoma who died of severe pulmonary fibrosis 81 days after the administration of high-dose BCNU (600 mg/m2). Thoracoscopic wedge resection of left upper lung performed 10 days before patient's death showed severe pulmonary fibrosis with prominent hyperplasia of alveolar macrophages and type II pneumocytes. We further used immunohistochemistry (IHC) to examine the relative role of platelet-derived growth factor-B (PDGF-B), insulin-like growth factor I (IGF-I), transforming growth factor-beta1 (TGF-beta1) and cyclooxygenase-2 (COX-2) in the pathogenesis of BCNU-related pulmonary fibrosis. Strong expressions of PDGF-B and IGF-1 on alveolar macrophages and type II pneumocytes were clearly demonstrated, but in contrast, the expressions of TGF-beta1 and COX-2 were almost undetectable. In conclusion, pulmonary fibrosis can develop early and progress rapidly after the administration of high-dose BCNU. The markedly increased expression of fibrogenic factors PDGF-B and IGF-1 on hyperplastic alveolar macrophages and hyperplastic type II pneumocytes may play an important role in the fibrogenesis of this disease. These novel findings may offer specific therapeutic targets in the treatment of BCNU-associated pulmonary fibrosis.

Significance of local electrostatic interactions in staphylococcal nuclease studied by site-directed mutagenesis.

In this paper, we show that amino acids Glu(73) and Asp(77) of staphylococcal nuclease cooperate unequally with Glu(75) to stabilize its structure located between the C-terminal helix and beta-barrel of the protein. Amino acid substitutions E73G and D77G cause losses of the catalytic efficiency of 24 and 16% and cause thermal stability losses of 22 and 26%, respectively, in comparison with the wild type (WT) protein. However, these changes do not significantly change global and local secondary structures, based on measurements of fluorescence and CD(222 nm). Furthermore, x-ray diffraction analysis of the E75G protein shows that the overall structure of mutant and WT proteins is similar. However, this mutation does cause a loss of essential hydrogen bonding and charge interactions between Glu(75) and Lys(9), Tyr(93), and His(121). In experiments using double point mutations, E73G/D77G, E73G/E75G, and E75G/D77G, significant changes are seen in all mutants in comparison with WT protein as measured by fluorescence and CD spectroscopy. The losses of thermal stability are 47, 59, and 58%, for E73G/D77G, E73G/E75G, and E75G/D77G, respectively. The triple mutant, E73G/E75G/D77G, results in fluorescence intensity and CD(222 nm) close to those of the denatured state and in a thermal stability loss of 65% relative to the WT protein. Based on these results, we propose a model in which significant electrostatic interactions result in the formation of a locally stable structure in staphylococcal nuclease.

Backbone dynamics of Escherichia coli thioesterase/protease I: evidence of a flexible active-site environment for a serine protease.

Escherichia coli thioesterase/protease I (TEP-I) is a member of a novel subclass of the lipolytic enzymes with a distinctive GDSLS motif. In addition to possessing thioesterase and protease activities, TEP-I also exhibits arylesterase activity. We have determined the (15)N nuclear magnetic spin relaxation rates, R(1) and R(2), and the steady state (1)H-(15)N heteronuclear Overhauser effect, measured at both 11.74 T and 14.09 T, of (u-(15)N) TEP-I. These data were analyzed using model-free formalism (with axially symmetric rotational diffusion anisotropy) to extract the backbone dynamics of TEP-I. The results reveal that the core structure of the central beta-sheet and the long alpha-helices are rigid, while the binding pocket appears to be rather flexible. The rigid core serves as a scaffold to anchor the essential loops, which form the binding pocket. The most flexible residues display large amplitude fast (ps/ns time-scale) motion and lie on one stripe whose orientation is presumed to be the ligand-binding orientation. We also detected the presence of several residues displaying slow (microseconds/ms time-scale) conformational exchanging processes. These residues lie around the binding pocket and are oriented perpendicularly to the orientation of the flexible stripe. Two of the putative catalytic triads, Ser10 and His157, and their neighbors show motion on the microseconds/ms time-scale, suggesting that their slow motion may have a role in catalysis, in addition to their possible roles in ligand binding. The presence of a flexible substrate-binding pocket may also facilitate binding to a wide range of substrates and confer the versatile functional property of this protein.

Crystal structures of the chromosomal proteins Sso7d/Sac7d bound to DNA containing T-G mismatched base-pairs.

Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. The crystal structures of Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 A, 2.2 A and 2.2 A, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation.

Crystallization of agglutinin from the seeds of Abrus precatorius.

Agglutinin protein purified from the seeds of Abrus precatorius has a high antitumour activity and was crystallized at room temperature with polyethylene glycol 8000 as the precipitant. The agglutinin crystal diffracted to 3.45 A and belongs to one of two possible tetragonal space groups, P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 141.91, c = 105.63 A. The asymmetric unit contains a heterotetrameric protein molecule of molecular weight 134 kDa and has a solvent content of approximately 38%.

Three-dimensional modelling of the catalytic domain of Streptococcus mutans glucosyltransferase GtfB.

Glucosyltransferases (GtfB/C/D) of Streptococcus mutans, a pathogen for human dental caries, synthesize water-insoluble glucan through the hydrolysis of sucrose. Genetic and biochemical approaches have identified several active sites of these enzymes, but no three-dimensional (3D) structural evidence is yet available to elucidate the subdomain arrangement and molecular mechanism of catalysis. Based on a combined sequence and secondary structure alignment against known crystal structures of segments from closely related proteins, we propose here the 3D model of an N-terminal domain essential for the sucrose binding and splitting in GtfB. A Tim-barrel of (alpha/beta)(8) structural characteristics is revealed and the structural correlation for two peptides is described.

Crystallization and preliminary X-ray crystallographic analysis of thioesterase I from Escherichia coli.

The Escherichia coli thioesterase I specifically catalyzes the deacylation of fatty acyl-CoA thioesters, especially those with long acyl groups (C(12)-C(18)). Single crystals of thioesterase I (E.C. 3. 1.2.2) from E. coli have been obtained using methoxypolyethylene glycol 5000 (PEG-MME 5K) as a precipitant at room temperature in 21 d. The crystals belong to the tetragonal space group P4(1)2(1)2 or its enantiomorph P4(3)2(1)2, with unit-cell parameters a = b = 50.85 (7), c = 171.5 (1) A. The crystals diffract to beyond 2.4 A resolution. There is one molecule of molecular weight 20.5 kDa in the asymmetric unit, with a solvent content of 55%.

Structure determination of porcine haemoglobin.

To investigate a potential candidate material for making artificial red blood cells to supplement blood transfusion, the X-ray structure of porcine haemoglobin at 1.8 A resolution was determined as part of research towards synthesizing human blood. Porcine haemoglobin was crystallized by the vapor-diffusion method, producing crystals of dimensions 0.3-0.5 mm after successive seeding. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 68.10, b = 72.27, c = 114.85 A. The initial phase was determined by the molecular-replacement method, using human oxyhaemoglobin as a model. The final R factor was 21.1% for 36 820 reflections after validation of 574 water molecules. The r.m.s. deviations of bond lengths, angles, torsion angles and improper angles from their ideal values are 0.017 A, 3.0, 20.6 and 1.8 degrees, respectively. The average B factor is 33.63 A(2) for the haemoglobin molecule and 50.53 A(2) for the water molecules. The structure could be superimposed on a 2.8 A resolution structure with an r.m.s. difference of 0.59 A in main-chain atomic positions and 1. 27 A in side-chain atomic positions. Porcine and human haemoglobins are compared. A tentative model for artificial blood is proposed based on the complementarity relationship of the surface charges between haemoglobin and the surrounding cell membrane.

Molecular structure of two crystal forms of cyclic triadenylic acid at 1A resolution.

The three dimensional structures of cyclic deoxytriadenylic acid, c-d(ApApAp), from two different trigonal crystal forms (space groups P3 and R32) have been determined by x-ray diffraction analysis at 1A resolution. Both structures were solved by direct methods and refined by anisotropic least squares refinement to R-factors of 0.109 and 0.137 for the P3 and R32 forms, respectively. In both crystal forms, each of the two independent c-d(ApApAp) molecules sits on the crystallographic 3-fold axis. All four independent c-d(ApApAp) molecules have similar backbone conformations. The deoxyriboses are in the S-type pucker with pseudorotation angles ranging from 156.7 degrees to 168.6 degrees and the bases have anti glycosyl torsion angles (chi falling in two ranges, one at -104.3 degrees and the other ranging from -141.0 degrees to -143.8 degrees). In the R32 form, a hexahydrated cobalt(II) ion is found to coordinate through bridging water molecules to N1, N3, and N7 atoms of three adjacent adenines and oxygen atoms of phosphates. Comparison with other structures of cyclic oligonucleotides indicates that the sugar adopts N-type pucker in cyclic dinucleotides and S-type pucker in cyclic trinucleotides, regardless whether the sugar is a ribose or a deoxyribose.

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala.

The 3-D structure of subtilisin YaB was computer modelled using the structures of subtilisin BPN', subtilisin Carlsberg and thermitase as templates. Gly124 and Gly151 located on both sides of the waist of the S1 pocket were selected for site-directed mutagenesis based on the modelled structure. The mutated ale genes coding for the mutant subtilisin YaB were expressed in Bacillus subtilis DB104. All of the G124 and G151 series of mutants exhibited an increase of relative catalytic activity for elastin-orcein against casein and myofibrillar proteins. The S1 substrate specificity of G124A, G124V and G151A mutants were assessed using various carbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro or Val)-(Ala, Phe or Leu)-p-nitroanilide [AA(P/V) (A/F/L)]. While G124A and G124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzed Ala, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPF and AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Km values approximately 3-10-fold higher than those of the wild-type enzyme. The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVA with kcat/Km values approximately 1-4-fold higher than those of the wild-type enzyme. These results clearly indicate that the S1 substrate specificity of G124A and G124V mutants was restricted to Ala and Gly, and G151A mutant to Ala, Gly and Leu.

Characterization of chicken-liver glutathione S-transferase (GST) A1-1 and A2-2 isoenzymes and their site-directed mutants heterologously expressed in Escherichia coli: identification of Lys-15 and Ser-208 on cGSTA1-1 as residues interacting with ethacrynic acid.

Escherichia coli-expressed chicken-liver glutathione S-transferase, cGSTA1-1, displays high ethacrynic acid (EA)-conjugating activity. Molecular modelling of cGSTA1-1 with EA in the substrate binding site reveals that the side chain of Phe-111 protrudes into the substrate binding site and possibly interacts with EA. Replacement of Phe-111 with alanine resulted in an enzyme (F111A mutant) with a 4.5-fold increase in EA-conjugating activity (9.2 mmol/min per mg), and an incremental Gibbs free energy (DeltaDeltaG) of 4.0 kJ/mol lower than that of the wild-type cGSTA1-1. Two other amino acid residues that possibly interact with EA are Ser-208 and Lys-15. Substitution of Ser-208 with methionine generated a cGSTA1-1(F111AS208M) double mutant that has low EA-conjugating activity (2.0 mmol/min per mg) and an incremental Gibbs free energy of +3.9 kJ/mol greater than the cGSTA1-1(F111A) single mutant. The cGSTA1-1(F111A) mutant, with an additional Lys-15-to-leucine substitution, lost 90% of the EA-conjugating activity (0.55 mmol/min per mg). The Km values of the cGSTA1-1(F111A) and cGSTA1-1(F111AK15L) mutants for EA are nearly identical. The wild-type cGSTA2-2 isoenzyme has a low EA-conjugating activity (0.56 mmol/min per mg). The kcat of this reaction can be increased 2. 5-fold by substituting Arg-15 and Glu-104 with lysine and glycine respectively. The KmEA of the cGSTA2-2(R15KE104G) double mutant is nearly identical with that of the wild-type enzyme. Another double mutant, cGSTA2-2(E104GL208S), has a KmEA that is 3.3-fold lower and a kcat that is 1.8-fold higher than that of the wild-type enzyme. These results, taken together, illustrate the interactions of Lys-15 and Ser-208 on cGSTA1-1 with EA.

Synthesis and cytotoxic evaluation of substituted sulfonyl-N-hydroxyguanidine derivatives as potential antitumor agents.

A series of sulfonyl-N-hydroxyguanidine derivatives was designed and synthesized for cytotoxic evaluation as potential anticancer agents on the basis of the lead compound LY-181984. Replacement of the ureido moiety of the lead compound with hydroxyguanidine provided a stable cytotoxic agent. The conformation of sulfonyl-N-hydroxyguanidine derivatives, such as N-(4-chlorophenyl)-N'-[(benzo[2,1,3]thiadiazol-4-yl)sulfonyl]-N"- hydroxyguanidine (4g), investigated utilizing HMBC NMR, theoretical calculations, and X-ray crystallography, indicated stacking of the two aromatic rings. The derivatives were evaluated for in vitro cytoxicity against five human tumor cell lines, including HepG2, TSGH 8302, COLO 205, KB, and MOLT-4. The cytotoxic activities of the derived compounds against the human tumor cell lines were equal to or greater than that of the lead compound. N-(4-Chlorophenyl)-N'-[[3,5-dichloro-4-(4-nitrophenoxy)phenyl]sulfonyl]- N"- hydroxyguanidine (4n) and N-(4-chlorophenyl)-N'-[[3,5-dichloro-4-(2-chloro-4-nitrophenoxy)phenyl] sulfonyl]-N"-hydroxyguanidine (4o) exhibited the greatest growth inhibition of solid tumor cell lines. Compound 4o was found to possess antitumor activity against murine K1735/M2 melanoma xenografts.

Identification of amino acid residues of abrin-a A chain is essential for catalysis and reassociation with abrin-a B chain by site-directed mutagenesis.

Abrin is a toxic protein consisting of two subunits, an enzymatic A chain (ABRaA) and a lectin-active B chain (ABRaB), linked by a disulfide bond. Site-directed mutagenesis was performed using PCR to study how the conserved amino acid residues, Tyr74, Tyr113, Glu164 and Trp198, around the active site of ABRaA are involved in enzyme catalysis, enzyme-substrate recognition and reassociation of ABRaA with ABRaB. The protein biosynthesis inhibitory activities of Y74F, Y113F and W198F were decreased moderately to that of wild type reABRaA, while that of E164Q decreased dramatically. Kinetic analysis showed that the kat of Y74F, Y113F and W198F resembled that of wild type, while the Km increased significantly. W198F did not reassociate with ABRaB to form heterodimers, while Y74F, Y113F and E164Q did. SDS-PAGE analysis of ABRaA treated with trypsin showed that reABRaA, Y74F, Y113F and E164Q survived digestion, whereas W198F was not protected from digestion. CD spectra revealed that W198F showed significant conformational changes. These observations suggest that E164 is directly involved in catalysis, and Tyr74, Tyr113 and Trp198 in substrate binding, while Trp198 also plays an important role in maintaining the conformation of ABRaA required for its reassociation with ABRaB.

Probing the domain structure of abrin-a by tryptic digestion.

Abrin-a is a potent plant toxin that consists of A and B chains linked by a disulfide bond. The abrin-a A chain (AaTA) has N-glycosidase activity while the abrin-a B chain (AaTB) has galactose-binding activity. By partial tryptic digestion, the domain structure of abrin-a was investigated. Seven tryptic fragments with molecular masses greater than 3500 Da were isolated and characterized. One fragment, designated T-21 and consisting of 153 amino acid residues, contained the major part of the second domain of AaTB and, after cross-linking of T-21 with glutaraldehyde, the reaction product had the same level of hemagglutinating activity as native abrin. When the T-21 fragment was conjugated with AaTA, the conjugate inhibited protein biosynthesis in HeLa cells. This suggests that the T-21 fragment is able to bind specifically to cells; its conjugate facilitates membrane translocation of AaTA into cells and consequently inhibits protein biosynthesis. T-21, with a molecular mass less than AaTB, is therefore a potentially useful substance for the preparation of immunotoxins.

Crystallization and preliminary X-ray analysis of chicken-liver glutathione S-transferase CL 3-3.

Five different crystal forms of recombinant chicken-liver glutathione S-transferase CL 3-3 have been obtained by the vapor-diffusion method. The form A crystals are monoclinic C2, a = 125.56, b = 85.81, c = 52.71 A and beta = 114.64 degrees, and diffract to 4 A resolution. The form B crystals are monoclinic P2(1), a = 105.13, b = 118.54, c = 62.62 A and beta = 124.74 degrees, and diffract to 2.8 A resolution. The form C crystals are orthorhombic C222(l), a = 101.69, b = 115.46, c = 95.40 A, and diffract to 2.8 A resolution. The form D crystals are tetragonal, P4(1)2(1)2 or P4(3)2(1)2, a = b = 115.31, c = 171.20 A and diffract to 3.5 A resolution. The form E crystals are hexagonal, P6(1) or P6(5), a = b = 104.23, c = 114.35 A, diffract to 3.5 A resolution. Forms A, C and E have one dimer of molecular weight 50 kDa, while forms B and D have two dimers per asymmetric unit, respectively.

Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides.

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103-108]. Here we describe protocols developed to purify polypeptides alpha and beta together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0 x 10(5) units per mg of protein. The respective denatured molecular masses of polypeptides alpha and beta were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 microM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.

Crystallization and preliminary x-ray analysis of volvatoxin A2 from Volvariella volvacea.

Volvatoxin A2, an ion channel disturbed cardiotoxic and hemolytic protein from the edible mushroom, Volvarilla volvacea, has been crystallized by the vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in sodium acetate buffer pH 4.6. The best crystals belong to the monoclinic space group C2 with unit cell dimensions a = 155.25 angstroms, b = 58.06 angstroms, c = 116.92 angstroms, and beta = 119.5 degrees. These crystals diffract to at least 2.2 angstroms and there are four molecules of molecular weight 24 kDa per asymmetric unit with a solvent content of 48%.

Selective contact during TCR recognition.

Recent structural analysis of the peptide-MHC complex reveals that an antigenic peptide binds to MHC in only one conformation and that side chains anchoring in the binding pocket would not contact TCR. The identification of all the MHC-anchoring residues on an antigenic peptide is a prerequisite to understand how a given peptide interacts with the TCR. In a combination of binding analysis and model simulation, model peptide lambda repressor cl 16-26 was shown to bind to I-Ek through four anchor residues (Leu18, IIe21, Glu23 and Lys26), a pattern found in many I-Ek-binding peptides. TCR reactivity analysis clearly indicates a great variation in the interaction with cl 16-26 by T cells generated from different strains of I-Ek-bearing mice. Most of the T cell generated from A/J mice reacted with the central regions of cl 16-26, while there is a great diversity on the recognition of cl 16-26 by T cells from C3H and B10.BR mice. Despite the diverse interactions with antigenic peptide by these T cells, most TCR-E-k contacts are limited to the central region of the I-Ek beta-chain. T cells recognizing only the N-terminal part of cl 16-26 were found to contact I-Ek at nearly the same residues as T cells interacting with the C-terminal of cl 16-26. TCR-I-Ek recognition was apparently independent of TCR-cl 16-26 contact. The discordant TCR-peptide and TCR-MHC interaction may represent a unique feature of TCR recognition.

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Vibrio mimicus.

Single crystals of arylesterase (EC 3.1.1.2) from Vibrio mimicus have been obtained from ammonium sulfate as a precipitant at room temperature for 2 months. The present crystals diffract up to 2.2 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 55.65(1) A, b = 53.46(1) A, c = 65.79(1) A, and beta = 106.54(1) degrees. There are two molecules of molecular weight 22 kDa in an asymmetric unit with a solvent content of 43%.