[Prediction of output fraction from single layer to commingling oil well by high performance liquid chromatography(HPLC)].

For crude oil from the same source, there is no obvious difference in chromatographic fingerprint of total hydrocarbons. But an evident difference in the concentrations of the series compounds of naphthalene and phenanthrene in oils has been found. Based on the fact, a new method for determine production allocation in commingled production, has been proposed. In this method, the series compounds of naphthalene and phenanthrene are selected as geochemistry indicator and are pretreated with aluminum oxide-silica gel column, and their relative concentration is determined by HPLC. Being verified by artificially mixing samples, the method can be used for the oils that can not be differentiated with the chromatographic fingerprint of total hydrocarbons, and error between predicted value and real value is 8% and its accuracy is better than that of the gas chromatographic fingerprint method. Based on the mixture principle, a model for prediction of output fraction of single oil layer is proposed. This model has been applied to production allocation calculation.

HPRT mutant T-cell lines from multiple sclerosis patients recognize myelin proteolipid protein peptides.

Mutation of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in a T-cell is believed to be an indication that the T-cell has been activated and has proliferated in vivo. HPRT mutant T-cell lines were generated from peripheral blood mononuclear cells from patients with MS and control subjects. More lines were isolated from the MS patients than from the control subjects. Using stringent criteria for recognition, none of the lines from MS-affected or control subjects recognized intact myelin basic protein (MBP) or myelin proteolipid protein (PLP) molecules. Using stringent criteria, two of the 10 MS patients harbored mutant lines each recognizing distinct PLP peptides (PLP peptide 40-60 recognized by 3 lines from one patient and PLP peptide 178-191 recognized by 2 lines from the other patient). A single line recognizing PLP peptide 89-106 was derived from 1 of 7 normal controls. HPRT mutant lines recognizing multiple epitopes of PLP which spanned much of the molecule could be isolated from MS patients, and to a lesser extent, normal subjects.

Substitutions in the HLA-DR alpha chain differentially affect DR7-restricted T-cell recognition of rabies virus antigen.

To investigate the functional roles of DR alpha residues in T-cell recognition, 20 mutants of the DR alpha chain were constructed by site-directed mutagenesis. These DR alpha mutants were expressed with WT DR(beta 1*0701) on mouse L cells and used as APC for four DR7-restricted T-cell clones specific for rabies virus antigens. The results indicate that the DR alpha residues are differentially involved in recognition of rabies virus antigen by different T-cell clones. Mutations in the floor of the antigen-binding groove (positions 9, 11, 22, and 24), on the alpha-helix (47, 55, 65, 66, and 72), and surprisingly on the outer loop (15, 18, and 19), abrogated recognition by at least one T-cell clone. Most of these residues appear to be involved in either peptide or TCR contact, based on the DR1 crystal structure. The involvement in T-cell recognition of DR alpha residues located in the outer loop outside the binding groove suggests that these residues may directly contact TCR, or indirectly contribute to the conformation of peptide sitting in the groove.

Differential effects of CTLA-4 substitutions on the binding of human CD80 (B7-1) and CD86 (B7-2).

CTLA-4 expressed on activated T cells binds to CD80 (B7-1) and CD86 (B7-2) molecules present on APC with high avidity and appears to deliver a negative regulatory signal to the T cell. We have investigated the kinetics of CTLA-4 binding to CD80 and CD86, together with the effects of selected CTLA-4 mutations on binding activity. The dissociation constants (Kd) for binding of CTLA-4-Ig to CD80 and CD86 transfectants were 8.1 and 6.7 nM, respectively. Surface plasmon resonance was used to determine kinetic parameters of CTLA-4-Ig binding to CD80-Ig and CD86-Ig fusion proteins and revealed enhanced association (ka) and dissociation (kd) rate constants for CD86-Ig compared with CD80-Ig. Furthermore, CD80-Ig and CD86-Ig fusion molecules demonstrated variable abilities to cross-compete for binding to several modified forms of CTLA-4-Ig. Differential binding of CD80 and CD86 to CTLA-4 was further revealed by analysis of 10 discrete CTLA-4 mutants. Five single amino acid substitutions within the CTLA-4 MYPPPY domain exerted modest effects on CD80 binding, but each of these substitutions completely abrogated CD86 binding. In addition, substitutions just N-terminal of the MYPPPY region, and within the CDR1-like region of CTLA-4, eliminated both CD80 and CD86 binding. Hence, CD80 and CD86 bind with different association/dissociation kinetics to similar, but distinct, sites on CTLA-4.

HLA-DR residues accessible under the peptide-binding groove contribute to polymorphic antibody epitopes.

Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the alpha-helix of DR beta chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DR beta chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR beta 1 and beta 2 domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(beta 1*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(beta 1*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR 16-DR7M and TAL13.1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the beta 2 domain, beta 180 and beta 181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptide-binding groove contribute to polymorphic antibody-binding epitopes.

Pocket 4 of the HLA-DR(alpha,beta 1*0401) molecule is a major determinant of T cells recognition of peptide.

To investigate the functional roles of individual HLA-DR residues in T cell recognition, transfectants expressing wild-type or mutant DR(alpha,beta 1*0401) molecules with single amino acid substitutions at 14 polymorphic positions of the DR beta 1*0401 chain or 19 positions of the DR alpha chain were used as antigen-presenting cells for five T cell clones specific for the influenza hemagglutinin peptide, HA307-19. Of the six polymorphic positions in the DR beta floor that were examined, mutations at only two positions eliminated T cell recognition: positions 13 (four clones) and 28 (one clone). In contrast, individual mutations at DR beta positions 70, 71, 78, and 86 on the alpha helix eliminated recognition by each of the clones, and mutations at positions 74 and 67 eliminated recognition by four and two clones, respectively. Most of the DR alpha mutations had minimal or no effect on most of the clones, although one clone was very sensitive to changes in the DR alpha chain, with loss of recognition in response to 10 mutants. Mutants that abrogated recognition by all of the clones were assessed for peptide binding, and only the beta 86 mutation drastically decreased peptide binding. Single amino acid substitutions at polymorphic positions in the central part of the DR beta alpha helix disrupted T cell recognition much more frequently than substitutions in the floor, suggesting that DR beta residues on the alpha helix make relatively greater contributions than those in the floor to the ability of the DR(alpha,beta 1*0401) molecule to present HA307-19. The data indicate that DR beta residues 13, 70, 71, 74, and 78, which are located in pocket 4 of the peptide binding site in the crystal structure of the DR1 molecule, exert a major and disproportionate influence on the outcome of T cell recognition, compared with other polymorphic residues.

Individual effects of the DR11-variable beta-chain residues 67, 71, and 86 upon DR(alpha,beta 1*1101)-restricted, peptide-specific T cell proliferation.

The four members of the HLA-DR11 family of class II molecules vary only by three or fewer amino acids via dimorphisms among DR beta-chain residues 67, 71, and 86. However, they differ markedly in their abilities to induce proliferation of DR(alpha,beta 1*1101)-restricted, peptide-specific T cell clones. To dissect which DR11-variable residues, individually and in combination, mediate these functional differences, we used as APC transfectants expressing DR molecules with one of all possible permutations of DR11-variable sequences, including the four DR11 family members, and four additional DR11 variant mutants. The abilities of the wild-type or mutant molecules to present two distinct influenza peptide Ags, HA307-19 and HA128-45, to T cells was assessed in in vitro T cell proliferation assays. Of the naturally dimorphic DR11 positions, residue beta 71 variation significantly influenced the ability of DR11 molecules to present both peptides to DR(alpha,beta 1*1101)-restricted T cells. Residue beta 86 variation had relatively less influence than reported in several other DR and peptide systems. Residue beta 67 variation usually appeared irrelevant to T cell proliferation, but in two mutants led to unexpected T cell proliferation independent of nominal peptide Ag. Peptide binding, assessed by flow cytometry, was not found to be altered by any mutations that disrupted DR(alpha,beta 1*1101)-like presentation. These data indicate that residue beta 71 exerts a central role in influencing the functional differences among DR11 molecules, whereas the widely studied dimorphism of residue beta 86 is not as generally influential in DR11 as in other alleles.

T cell receptor and peptide-contacting residues in the HLA-DR17(3) beta 1 chain.

Previously, we have proposed that the beta 1 residues 9-13, 26, 28 and 86 in HLA-DR17, the most common subtype of DR3, might be critical for the binding of an immunodominant, mycobacterial epitope (peptide 3-13 of the 65-kDa heat shock protein). In order to examine directly (i) which DR17 residues are involved in peptide binding, (ii) whether the same or other DR17 residues are involved in the binding of different peptides, and (iii) whether subtle differences in the mode of peptide binding can influence T cell stimulation, we have now systematically mutated 15 highly polymorphic DR17 beta 1 residues, located in the proposed peptide binding groove of DR17, and examined the effect thereof on binding and presentation of two peptides, hsp65 p3-13 and p56-65 of the 30/31-kDa secreted mycobacterial protein. Mutations in residues 28 (D-->H) and 86 (V-->G) completely eliminated binding of p3-13 and significantly reduced binding of p56-65. A mutation in residue 26 (Y-->F) decreased binding of p3-13 but did not affect binding of p56-65. Substitutions of amino acid residues 28, 67, 71 and 86 in the DR17 beta 1 chain abrogated peptide-specific stimulation of both the p3-13- and the p56-65-specific T cell clones, while specific stimulation by only one peptide was eliminated by substitution at positions 26 and 74 (p3-13) and by substitution of residues 11 and 37 (p56-65). The observation that substitution of several other peptide-contacting DR17 beta 1 chain residues does not significantly affect peptide binding but does affect T cell stimulation, suggests that these substitutions alter the conformation of the bound peptide.

Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes.

The contributions of individual amino acids in the polymorphic beta chain and the conserved alpha chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the beta 1 domain and 11 positions in the alpha 1 domain. The beta 1-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR alpha 1 domain were based on the sequences of other human and mouse class II alpha chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the beta 1-domain alpha-helix caused substantial reduction in the responses of all of the clones. Substitutions at multiple positions in the beta 1-domain floor and in the alpha 1 domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the beta 1 domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules.

Analysis of monoclonal antibodies specific for unique and shared determinants on HLA-DR4 molecules.

The specificities for seven mAbs to HLA-DR4 were determined initially using homozygous BCLs and L-cell transfectants expressing wild-type DR molecules. Three antibodies (NFLD.D1, NFLD.M1, and NFLD.D7) bound all DR4 molecules, but only one was specific for DR4. Four antibodies (NFLD.D2, NFLD.D3, NFLD.D8, and NFLD.D10) reacted with some but not all DR4 subtypes and had extra reactions, particularly with DR gene products associated with susceptibility to RA. To localize the antibody-binding epitopes on DR4 molecules, the antibodies were then analyzed on transfectants expressing hybrid genes, which were generated by exon shuffling of DRB1*0403 and DRB1*0701. Two of the pan-DR4 antibodies bound epitopes that require the beta 2 domain while the third mapped primarily to the HVR-I region. One antibody NFLD.D10 to subtypes of DR4 mapped to residues 40-97 on DR beta 1*0403 chains. Comparison of reaction patterns with amino acid sequences suggest that the antibodies against subtypes of DR4 are specific primarily for a region containing sequences postulated to determine susceptibility to RA.

HLA-DR alpha chain residues located on the outer loops are involved in nonpolymorphic and polymorphic antibody-binding epitopes.

The structure-function relationships of the HLA-DR alpha chain have been analyzed by identifying DR alpha residues involved in several nonpolymorphic and polymorphic antibody epitopes. Antibody binding to transfectants expressing a WT or mutant DR alpha chain with the WT DR(beta 1*0701) chain was analyzed. Our results indicate that residues 18, 36, and 39 located on the outer loops of the DR alpha chain are critical for one or more of the epitopes recognized by the SG157, Q2/70, L243, LB3.1, D1-12, and CL413 mAbs. Similar results were obtained when the DR alpha position 18 and 39 mutants were expressed with other DR beta 1 alleles. Furthermore, residues 15 and 18 of the DR alpha chain were shown to be involved in the epitopes of two polymorphic mAbs, HU-26 and I-2, whose epitopes also include residue 4 of the corresponding DR beta chains. In addition to their involvement in antibody-binding epitopes, residues in this region on the outer surface of the DR alpha chain have also been shown to be involved in superantigen binding and presentation and T-cell recognition of foreign antigen, emphasizing the functional importance of DR alpha-chain residues located outside of the peptide-binding groove.

HLA-DR beta chain residues that are predicted to be located in the floor of the peptide-binding groove contribute to antibody-binding epitopes.

The purpose of this study was to identify polymorphic residues in HLA-DR beta chains that are involved in the epitopes recognized by DRw52-like mAbs. Flow cytometric analysis of antibody binding to mouse fibroblast transfectants expressing wild-type or mutant DRw52-associated DR beta chains demonstrated that the amino acid at position 77, which is predicted to be on a solvent-accessible surface of the alpha-helix, contributes to the epitopes recognized by the 7.3.19.1 and 21r5 mAbs. In contrast, residues that are not predicted to occupy accessible positions on the alpha-helix contribute to binding of the I-LR2 and UL-52 mAbs. Substitutions at positions 10, 12, and 51, but not 9, 11, and 13, affect these epitopes. It is interesting that antibody binding is influenced by amino acid substitutions at DR beta positions 10 and 12 because the class II model predicts that residues at these positions are located in the middle of the floor of the peptide-binding site, with their side chains pointing down. These findings emphasize the functional importance of polymorphic residues whose side chains are not predicted to point into or up from the antigen-binding site and they raise the possibility that peptides contribute to the generation of these epitopes.

Identification of HLA-DR alpha chain residues critical for binding of the toxic shock syndrome toxin superantigen.

Staphylococcal toxic shock syndrome toxin 1 (TSST-1) binds to major histocompatibility complex class II molecules, and the toxin-class II complexes induce proliferation of T cells expressing V beta 2 sequences. To define the residues involved in TSST-1 binding, a set of transfectants expressing 21 HLA-DR alpha chain mutants were analyzed for their abilities to bind and present TSST-1 and to present an antigenic peptide. Mutations at DR alpha positions 36 and 39 markedly decreased the ability of the DR7 molecule to bind and present TSST-1 but did not affect the ability to present an antigenic peptide. These data indicate that DR alpha residues 36 and 39, predicted to be located on an outer loop, are important in the formation of the TSST-1 binding site on DR molecules.

Diverse locations of amino acids in HLA-DR beta chains involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0101), DR(alpha, beta 1*1101), and DR(alpha,beta 3*0202) molecules.

In a previous study, we used transfectants expressing hybrid HLA-DR(beta 1*0403)/DR(beta 1*0701) chains to map sequences involved in polymorphic antibody binding epitopes on DR(alpha, beta 1*0403) or DR(alpha, beta 1*0701) molecules. Amino acids 1-40 of the beta 1 domain were found to make the major contributions to most of the antibody binding epitopes studied. To begin to localize sequences that contribute to polymorphic antibody epitopes on DR(alpha,beta 1*0101), DR(alpha,beta 1*1101) and DR(alpha,beta 3*0202) molecules, we used indirect immunofluorescence and flow cytometry to assess the binding of mAb to transfectants expressing hybrid DR(beta 1*0101)/DR(beta 1*1101) or DR(beta 1*1101)/DR(beta 3*0202) chains that divide the DR beta chain into three segments: amino acids 1-40, 41-97, and the beta 2 domain. The results indicate that amino acids 41-97 of the beta 1 domain on DR(beta 1*0101), DR(beta 1*1101), or DR(beta 3*0202) are critical in most of the epitopes, including those recognized by human antibodies MP4 and MP12, and mouse mAb GS88.2, I-LR1, 21r5, and 7.3.19.1, whereas amino acids 1-40 of DR(beta 1*1101) are critical in the epitope recognized by the MCS-7 mAb, and both segments 1-40 and 41-97 of DR(beta 1*1101) are important in the epitopes recognized by the I-LR2 and UL-52 mAbs. Based on these data and comparison of DR beta allelic protein sequences, the residues that may play critical roles in these antibody binding epitopes are predicted.

Identification of residues involved in polymorphic antibody binding epitopes on HLA-DR molecules.

Based on previous studies it was predicted that amino acids 4 or 25 of the DR4 beta 1 and DR7 beta 1 chains are involved in polymorphic antibody binding epitopes on DR4 or DR7 molecules. These predictions were tested by analyzing monoclonal antibody (mAb) binding to transfectants expressing mutant DR4 beta 1 or DR7 beta 1 chains with single amino acid substitutions at positions 4 or 25. Antibody binding to transfectants expressing additional DR4/7 beta 1 hybrids was also analyzed to assess further the contributions of four segments of the DR4 beta 1 or DR7 beta 1 chains: amino acids 1-20, 21-40, 41-97, and the beta 2 domain. Single amino acid substitutions at positions 4 and 25 of the DR4 beta 1 chain or DR7 beta 1 chain eliminate binding of several mAb to DR4 or DR7 molecules, documenting that these residues are involved in antibody epitopes. However, the data with the hybrid DR4/7 beta 1 chains indicate that some of these epitopes require contributions from both segments 1-20 and 21-40 of these DR beta chains, whereas other epitopes can be generated by placing the appropriate segment in the context of the other DR beta chain. In addition, the data with other mAb indicate that their epitopes are determined primarily by sequences within the 41-97 segment or in the beta 2 domain.