Primary and tertiary structures of the Fab fragment of a monoclonal anti-E-selectin 7A9 antibody that inhibits neutrophil attachment to endothelial cells.

The murine monoclonal IgG1 antibody 7A9 binds specifically to the endothelial leukocyte adhesion molecule-1 (E-selectin), inhibiting the attachment of neutrophils to endothelial cells. The primary and three-dimensional structures of the Fab fragment of 7A9 are reported. The amino acid sequence was determined by automated Edman degradation analysis of proteolytic fragments of both the heavy and light chains of the Fab. The sequences of the two chains are consistent with that of the IgG1 class with an associated kappa light chain with two intrachain disulfide bridges in each of the heavy and light chains. The tertiary structure of the antibody fragment was determined by x-ray crystallographic methods at 2.8 A resolution. The F(ab')2 molecule, treated with dithiothreitol, crystallizes in the space group P2(1) 2(1) 2(1) with unit cell parameters a = 44.5 A, b = 83.8 A, and c = 132.5 A with one Fab molecule in the asymmetric unit. The structure was solved by the molecular replacement method and subsequently refined using simulated annealing followed by conventional least squares optimization of the coordinates. The resulting model has reasonable stereochemistry with an R factor of 0.195. The 7A9 Fab structure has an elbow bend of 162 degrees and is remarkably similar to that of the monoclonal anti-intercellular adhesion molecule-1 (ICAM-1) antibody Fab fragment. The 7A9 antigen combining site presents a groove resembling the structure of the anti-ICAM-1 antibody, and other antibodies raised against surface receptors and peptides. Residues from the six complementary determining regions (CDRs) and framework residues form the floor and walls of the groove that is approximately 22 A wide and 8 A deep and that is lined with many aromatic residues. The groove is large enough to accommodate the loop between beta-strands beta4 and beta5 of the lectin domain of E-selectin that has been implicated in neutrophil adhesion (1).

Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis.

Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.

Regeneration of antidigoxin binding activity in an antibody by changes surrounding the original binding defect.

A panel of antibodies which differ in their L chain structures and which bind to structurally defined haptens, would be useful in investigating L chain structure and function. In a previous study, chain recombinant antibody CR24 (26-10 H, 45-20 lambda) was produced by hybridoma-hybridoma fusion. Although both parental antibodies bound digoxin with high affinity, CR24 lacked detectable digoxin-binding activity. Hybridoma CR24 was subsequently fused with H chain-loss hybridomas in order to produce a panel of antibodies composed of 26-10 H chains and 26-10 "like" L chains. Two antibodies produced were CR260 which demonstrated digoxin-binding activity and CR256 which did not. CR260 and CR256 expressed only one amino acid difference (Pro to Leu at L-96). This difference resulted in the CR256 binding defect. In this report, two new antidigoxin antibodies are described. One, SR2E7, contained the Pro to Leu (L-96) defect, but still bound digoxin. Binding affinities and binding specificity patterns, as well as complete VL DNA sequence and corresponding protein sequence of the new digoxin binding antibody L chains (SR2E7 and SR1C7) are presented. Both kappa L chains are highly homologous to the 26-10 kappa L chain as well as the BALB/c germline gene K5.1. These results suggest that antibodies which are initially defective in binding activity can be cured by changing specific amino acids involved in determining the binding-site structure. Molecular modelling studies of the binding-site region were completed to address L chain structural changes induced by specific amino acid substitutions.

A study of intermediates involved in the folding pathway for recombinant human macrophage colony-stimulating factor (M-CSF): evidence for two distinct folding pathways.

The folding pathway for a 150-amino acid recombinant form of the dimeric cytokine human macrophage colony-stimulating factor (M-CSF) has been studied. All 14 cysteine residues in the biologically active homodimer are involved in disulfide linkages. The structural characteristics of folding intermediates blocked with iodoacetamide reveal a rapid formation of a small amount of a non-native dimeric intermediate species followed by a slow progression via both monomeric and dimeric intermediates to the native dimer. The transition from monomer to fully folded dimer is complete within 25 h at room temperature at pH 9.0. The blocked intermediates are stable under conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thus represent various dimeric and folded monomeric species of the protein with different numbers of disulfide bridges. Peptide mapping and electrospray ionization mass spectrometry revealed that a folded monomeric species of M-CSF contained three of the four native disulfide bridges, and this folded monomer also showed some biological activity in a cell-based assay. The results presented here strongly suggest that M-CSF can fold via two different pathways, one involving monomeric intermediates and another involving only dimeric intermediates.

Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock.

A quantitative sandwich ELISA for E-selectin in the fluid phase (soluble E-selectin, sEs) has been developed that is sensitive to 100 pg/ml. The assay shows no reactivity with either L- or P-selectins. We have used this to determine the fate of E-selectin after cell-surface expression and to test whether levels measured in vivo may represent the state of endothelial activation. E-selectin was first detectable in supernatants of IL-1-stimulated endothelial cells at 24 h, and increased slowly up until 72 h. However, over this time period the total E-selectin detectable in the system (cells plus supernatants) declined dramatically. 125I-surface-labeled endothelial cells cultured for 24 h show an E-selectin of reduced m.w. in the supernatant, indicating that the molecule is shed from the surface. The shed form also appears to be slightly smaller than the intact membrane form as determined from immunoprecipitation and molecular sieving studies. In addition, the cytoplasmic domain of the molecule found in supernatants of activated endothelial cells and in serum is not intact as determined by loss of reactivity with an antipeptide antibody specific for the cytoplasmic domain. We have examined the sera of 71 normal individuals. Without exception, sEs was found in serum in the range of 0.13 to 2.8 ng/ml, suggesting that even in the absence of overt inflammatory processes E-selectin is being synthesized and released into the bloodstream. In addition, bacteremic patients with hypotension, but not those without, showed markedly elevated sEs values. As determined by cell-binding studies, the blood-derived form of E-selectin is biologically active.

Utilization of an active serine 101----cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.

Thioesterase II is a 29-kDa monomer which, in certain specialized tissues, acts as a chain terminator in fatty acid synthesis by hydrolyzing medium-chain fatty acids from the fatty acid synthase. As with serine proteases, hydrolysis appears to involve acylation of the active site serine residue (Ser-101) assisted by a histidine, tentatively identified as His-237. To determine whether in the folded protein His-237 is close enough to accept a proton from the Ser-101 hydroxyl, we have made use of a Ser101Cys mutant which retains up to 90% of catalytic activity. Unlike the wild-type enzyme, the S101C thioesterase is inhibited with stoichiometric amounts of the bifunctional alkylating reagent 1,3-dibromopropanone. To facilitate identification of the alkylated residue(s), the keto group introduced into the dibromopropanone-modified S101C mutant was radiolabeled by reduction with sodium [3H] borohydride. The protein was then digested and the radiolabeled peptides analyzed by amino acid sequencing and mass spectrometry. The experimental data unambiguously showed that dibromopropanone cross-linked the active site Cys-101 with His-237, demonstrating that these residues are positioned within 5 A of each other. These data strongly support the hypothesis that in the wild-type thioesterase His-237 accepts a proton from Ser-101, thus increasing its nucleophilic character and improving the catalytic efficiency of the enzyme. The possibility that exchange of cysteine and serine active site residues has occurred in the evolution of thioesterases is discussed.

Structural organization of the multifunctional animal fatty-acid synthase.

The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/acetyl transferase, residues 1630-1850 to the enoyl reductase, residues 1870-2100 to the oxyreductase, residues 2114-2190 to the acyl-carrier protein and residues 2200-2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.

Cloning, sequencing, and characterization of Escherichia coli thioesterase II.

The gene (tesB) encoding Escherichia coli thioesterase II, a low-abundance enzyme of unknown physiological function which can hydrolyze a broad range of acyl-CoA thioesters, has been localized by transposon mutagenesis, cloned and sequenced. A two-cistron construct containing both the lac and tesB promoters was used successfully to overexpress the 286-residue polypeptide. The recombinant enzyme constituted up to 25% of the soluble proteins of E. coli and was readily purified to homogeneity as a tetramer of approximately 120,000 Da. Amino-terminal sequence analysis and electrospray ionization mass spectrometry confirmed the identity of the thioesterase and revealed that the amino-terminal formyl-methionine had been removed yielding a subunit species of average molecular mass 31,842 Da. The protein does not contain the GXSXG motif found characteristically in animal thioesterases which function as chain-terminating enzymes in fatty acid synthesis and exhibits no sequence similarity with these or any other known proteins. Activity of the recombinant enzyme was inhibited by iodoacetamide and diethylpyrocarbonate. The carboxamidomethylated residue was identified as histidine 58, and a role for this amino acid in catalysis is suggested. E. coli strains having a large deletion within the genomic tesB gene grew normally but retained a low level of thioesterase activity toward decanoyl-CoA. This residual activity indicates the presence of an additional decanoyl-CoA hydrolase in E. coli. Over-expression of the recombinant enzyme, under control of the lac promoter, did not alter the fatty acids synthesized by E. coli at any stage of cell growth and the physiological role of this enzyme remains an enigma.

Ustilago maydis virus P4 killer toxin: characterization, partial amino terminus sequence, and evidence for glycosylation.

The toxin from Ustilago maydis virus P4 was purified to homogeneity and characterized. The native molecular mass, using size-exclusion HPLC was estimated to be 7.2 kDa. The purified toxin was composed of a single subunit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reduced and nonreduced conditions resulted in estimated molecular masses of 8.4 and 7.4 kDa, respectively. The purified toxin was found to be glycosylated when tested for carbohydrates using the phenol-sulfuric acid method, Schiff's base reagent, and a Glycan detection kit and when probed against different biotinylated lectins. Partial amino acid sequence analysis of the purified toxin indicated a free N-terminus, 16% glycine, and 23% basic amino acid residues. No homology was found to either the alpha or the beta subunit of the toxin encoded by U. maydis infected with the P6 virus.

Biotinylated parathyroid hormone as a probe for the parathyroid hormone receptor. Structure-function analysis and detection of specific binding to cultured bone cells by flow cytometry.

The synthetic bovine parathyroid hormone (PTH) analog (Nle8, Nle18, Tyr34) bovine PTH(1-34)amide (bPTH(1-34)amide) was reacted with biotinyl-epsilon aminocaproic acid-N-hydroxysuccinimide under conditions which yielded five isoforms which were fractionated by a combination of reversed phase and ion-exchange chromatography. These reaction products were analyzed by automated Edman degradation in a manner which allowed us to specify the location and number of biotin residues on picomole quantities of hormone. The ability of each of these isoforms to induce a rise in intracellular cAMP in the ROS 17/2.8 cell line allowed us to evaluate the effect on function of biotinylation at different residues. Derivatized PTH molecules which contained a single biotin at either lysine 13, lysine 26, or lysine 27 possessed full biological activity. However, bioactivity was significantly reduced when position 13 plus either lysine 26 or 27 were biotinylated. Biological activity was lost when all 3 lysine residues were biotinylated. Biotinylation of the alpha-NH2 group of alanine at the NH2 terminus also resulted in a total loss of activity. Hence, unlike the effect of altering the alanine at position 1, modification of a single lysine residue at positions 13, 26, and 27 has a less critical effect on biological activity of the molecule. However, biotinylation of all three lysines results in a biologically inert PTH derivative and suggests that changes in isoelectric point, hydrophobicity, or tertiary structure may strongly influence hormone function. A fully bioactive-mixture of isoforms was used to detect receptors on ROS 17/2.8 cells by flow cytometry using fluorescein isothiocyanate-avidin as a fluorescent indicator. Binding to cell surface receptors was saturable and could be inhibited by native bPTH(1-34) but not by transforming growth factor beta, calcitonin or insulin. Moreover, PTH receptors could also be detected on primary cultures of human bone cells and human fibroblasts.

Purification and molecular properties of the toxin coded by Ustilago maydis virus P4.

The toxin from the P4 strain of Ustilago maydis was purified and characterized using a series of gel-filtration and ion-exchange columns. The apparent molecular weight of the purified toxin was estimated from gel electrophoresis to be 11.3 kd in the presence of 2-mercaptoethanol and 10.3 kd in the absence of 2-mercaptoethanol. Amino acid analysis indicated 12% basic amino acids, 14% acidic amino acids and 16% glycine. The toxin was also stable to filtration and repeated freezing at -20 degrees C and thawing.

Molecular cloning and sequencing of cDNAs encoding the entire rat fatty acid synthase.

Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.

Complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase from rat mammary gland.

The complete amino acid sequence of the medium-chain S-acyl fatty acid synthetase thio ester hydrolase (thioesterase II) from rat mammary gland is presented. Most of the sequence was derived by analysis of peptide fragments produced by cleavage at methionyl, glutamyl, lysyl, arginyl, and tryptophanyl residues. A small section of the sequence was deduced from a previously analyzed cDNA clone. The protein consists of 260 residues and has a blocked amino-terminal methionine and calculated Mr of 29,212. The carboxy-terminal sequence, verified by Edman degradation of the carboxy-terminal cyanogen bromide fragment and carboxypeptidase Y digestion of the intact thioesterase II, terminates with a serine residue and lacks three additional residues predicted by the cDNA sequence. The native enzyme contains three cysteine residues but no disulfide bridges. The active site serine residue is located at position 101. The rat mammary gland thioesterase II exhibits approximately 40% homology with a thioesterase from mallard uropygial gland, the sequence of which was recently determined by cDNA analysis [Poulose, A.J., Rogers, L., Cheesbrough, T. M., & Kolattukudy, P. E. (1985) J. Biol. Chem. 260, 15953-15958]. Thus the two enzymes may share similar structural features and a common evolutionary origin. The location of the active site in these thioesterases differs from that of other serine active site esterases; indeed, the enzymes do not exhibit any significant homology with other serine esterases, suggesting that they may constitute a separate new family of serine active site enzymes.

Amino acid sequence of the serine active-site region of the medium-chain S-acyl fatty acid synthetase thioester hydrolase from rat mammary gland.

Medium-chain S-acyl fatty acid synthetase thioester hydrolase (thioesterase II), a discrete monomeric enzyme of 29 kDa, regulates the product specificity of the de novo lipogenic systems in certain specialized mammalian and avian tissues, such as mammary and uropygial glands. The amino acid sequence of a 57-residue region containing the active site of the rat mammary gland enzyme has been established by a combination of amino acid and cDNA sequencing. Thioesterase II was radiolabeled with the serine esterase inhibitor [1,3-14C]diisopropyl-fluorophosphate and digested sequentially with cyanogen bromide, Staphylococcus aureus V8 protease and trypsin. A radiolabeled tryptic peptide was isolated and sequenced by automated Edman degradation and the location of the active-site residue established. The amino acid sequence was confirmed by sequencing an overlapping, unlabeled peptide, obtained by V8 digestion of the whole enzyme, and by dideoxynucleotide sequencing of a thioesterase II cDNA clone isolated from a lambda gt11 expression library. The active center contains the motif Gly-Xaa-Ser-Xaa-Gly, characteristic of the serine esterase family of enzymes. A seven-residue region around the essential serine of the rat mammary thioesterase II, Phe-Gly-Met-Ser-Phe-Gly-Ser, is completely homologous with a region of the mallard uropygial thioesterase, recently analyzed by cDNA sequencing, indicating that this is likely to be the active site of the avian enzyme. Overall homology between the mammalian and avian enzymes for the 57-amino-acid residue region is 47% and suggests that the two enzymes may share a common evolutionary origin.

Elevated G gamma gene expression with specific beta S gene haplotype, normal gamma gene maps and presence of the Xmn I site -158 5' to the G gamma gene in Indian sickle cell anemia.

In nine Indian patients ranging in age between four and 61 years, with mild Hb SS disease and very high Hb F levels, the G gamma globin chain levels of their fetal hemoglobin ranged between 64.0% and 70.0%, with a mean of 68.1% (S.D. +/- 2.6) of the total amount of gamma-globin chains. Eight of the nine patients were homozygous for a specific beta S gene haplotype #31. The other one was doubly heterozygous for the same specific haplotype and another haplotype, which differed from haplotype #31 by the presence of Bam HI site 3' to the beta gene and absence of Pvu II site 5' to the psi beta gene. The gamma gene organization studied by Pst I restriction enzyme analysis was found to be normal and the Xmn I site -158 5' to G gamma gene was present in all patients examined.

Mammalian fatty acid synthetase is a structurally and functionally symmetrical dimer.

We have explored a comprehensive experimental approach to determine whether the two condensing-enzyme active centers of the mammalian fatty acid synthetase are simultaneously functional. Our strategy involved utilization of trypsinized fatty acid synthetase, which is a nicked homodimer composed of two pairs of 125 + 95-kDa polypeptides. These core polypeptides lack the chain-terminating thioesterase domains but retain all other functional domains of the native enzyme and can assemble long-chain acyl moieties at a rate equal to that of the native enzyme. The 4'-phosphopantetheine content of these enzyme preparations, estimated from the amount of beta-alanine present, from the amount of taurine formed by performic acid oxidation and from the amount of carboxymethylcysteamine formed by alkylation with iodo[2-14C]acetate, was typically 0.86 mol/mol 95-kDa polypeptide. The stoichiometry of long-chain acyl-enzyme synthesis, measured with radiolabeled precursors, indicated that 0.84 mol acyl-chains were assembled/mol 95-kDa polypeptide. When the small amount of apoenzyme present is taken into account, this stoichiometry translates to 1.94 acyl chains per holoenzyme dimer. The 125-kDa polypeptide of one subunit could be cross-linked to the 95-kDa polypeptide of the other subunit by 1,3-dibromo-2-propanone yielding a single molecular species of 220 kDa. Cross-linking was accompanied by a loss of condensing-enzyme activity. This result is consistent with a structurally symmetrical model for the animal fatty acid synthetase [J.K. Stoops and S.J. Wakil (1981) J. Biol. Chem. 256, 5128-5133] in which the juxtaposed 4'-phosphopantetheine and cysteine thiols of opposing subunits that form the two potential catalytic centers for condensing activity are readily susceptible to cross-linking. Both half-maximal cross-linking and 50% inhibition of activity were observed with 1 mol 1,3-dibromo-2-propanone bound/mol enzyme. After assembly of long-chain acyl moieties on the 4'-phosphopantetheine residues, no vacant condensing-enzyme active sites were demonstrable either by cross-linking with 1,3-dibromo-2-propanone or by formation of carboxymethylcysteamine on treatment with iodoacetate. These results are consistent with a structurally and functionally symmetrical model for the mammalian fatty acid synthetase in which the two condensation sites are simultaneously active.

Human hemoglobin Portland II (zeta 2 beta 2). Isolation and characterization of Portland hemoglobin components and their constituent globin chains.

Two types of embryonic hemoglobins (Hb) containing zeta chains have been identified in the blood of several neonates of Chinese origin with homozygous alpha-thalassemia. In addition to Hb Portland I (zeta 2 gamma 2) which was previously reported, another embryonic hemoglobin has been detected and found to contain zeta chains and beta chains. It is being designated Hb Portland II and has the formula (zeta 2 beta 2). It has a mobility slightly slower than that of Hb A on starch gel electrophoresis at pH 8.6 and has been found in the hemolysates of blood of some but not all hydropic infants. Another component with a mobility faster than that of Hb A2 on starch gel has been isolated from the blood of some hydropic neonates. This latter component is postulated to be zeta 2 delta 2. The occurrence of Hb Portland I and Hb Portland II in these hydropic neonates is consistent with the hypothesis that, in the absence of normal alpha chain production, zeta chains are continued to be produced at later states of development than normal and form tetramers with each of the beta-like globin chains. Because Hb Portland II has not been found in blood from all hydropic neonates, we postulate that the presence of this hemoglobin in these fetuses may be correlated with the gestational age of the fetus at the time of birth.

Separation of zeta (zeta) and various gamma (gamma) chains of human embryonic hemoglobin Portland I by reverse-phase high-performance liquid chromatography.

The zeta (zeta) and various gamma (gamma) chains of Hb Portland I have been separated and isolated from blood samples obtained from neonates with hydrops fetalis due to homozygous alpha-thalassemia. By using developers containing acetonitrile, methanol, and potassium phosphate and either an analytical (3.9 mm x 30 cm) or a preparative (7.8 mm x 30 cm) muBondapak C-18 column (Waters), globin chains from 200 micrograms to 5.0 mg have been isolated in pure forms. Analytical and preparative procedures using short (50-min duration) and extended (186-min duration) gradient programs have been developed. In addition to the type of column and developer conditions, the following factors are found to be important: (a) preparation of sample, (b) sample loading, and (c) cleaning of the column. Preliminary studies indicate that the yield ranges from 40 to 60% depending on the type of globin sample and the age of the column. This procedure also permits the separation of alpha, beta, and various gamma globin chains from fetal and adult samples.