Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin.

BACKGROUND AND OBJECTIVE: Resistance of thrombi to plasmin digestion depends primarily on the amount of α(2)-antiplasmin (α(2)AP) incorporated within fibrin. Circulating prolyl-specific serine proteinase, antiplasmin-cleaving enzyme (APCE), a homologue of fibroblast activation protein (FAP), cleaves precursor Met-α(2)AP between -Pro12-Asn13- to yield Asn-α(2)AP, which is crosslinked to fibrin approximately 13× more rapidly than Met-α(2)AP and confers resistance to plasmin. We reasoned that an APCE inhibitor might decrease conversion of Met-α(2)AP to Asn-α(2)AP and thereby enhance endogenous fibrinolysis. METHODS AND RESULTS: We designed and synthesized several APCE inhibitors and assessed each vs. plasma dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP), which have amino acid sequence similarity with APCE. Acetyl-Arg-(8-amino-3,6-dioxaoctanoic acid)-D-Ala-L-boroPro selectively inhibited APCE vs. DPPIV, with an apparent K(i) of 5.7 nm vs. 6.1 µm, indicating that an approximately 1000-fold greater inhibitor concentration is required for DPPIV than for APCE. An apparent K(i) of 7.4 nm was found for POP inhibition, which is similar to 5.7 nm for APCE; however, the potential problem of overlapping FAP/APCE and POP inhibition was negated by our finding that normal human plasma lacks POP activity. The inhibitor construct caused a dose-dependent decrease of APCE-mediated Met-α(2)AP cleavage, which ultimately shortened plasminogen activator-induced plasma clot lysis times. Incubation of the inhibitor with human plasma for 22 h did not lessen its APCE inhibitory activity, with its IC(50) value in plasma remaining comparable to that in phosphate buffer. CONCLUSION: These data establish that inhibition of APCE might represent a therapeutic approach for enhancing thrombolytic activity.

Why alpha-antiplasmin must be converted to a derivative form for optimal function.

BACKGROUND: Human alpha(2)-antiplasmin (alpha(2)AP), the primary inhibitor of fibrinolysis, is secreted from the liver into plasma as a 464-residue protein with Met as the N-terminus. An R6W polymorphism has been suggested to affect fibrinolytic rate. Within circulating blood, antiplasmin-cleaving enzyme (APCE) cleaves Met-alpha(2)AP(R6) faster than Met-alpha(2)AP(W6) at the Pro12-Asn13 bond to yield Asn-alpha(2)AP. OBJECTIVES: To compare Met-alpha(2)AP(R6), Met-alpha(2)AP(W6) and Asn-alpha(2)AP for crosslinking with fibrin and the ability to protect fibrin from digestion by plasmin. METHODS AND RESULTS: Asn-alpha(2)AP utilizes Gln2 (Gln14 in Met-alpha(2)AP) to become crosslinked to fibrin approximately twelvefold faster than Met-alpha(2)AP(R6) or Met-alpha(2)AP(W6), and this enhances the resistance of fibrin to plasmin. All three forms of alpha(2)AP inhibit plasmin at identical rates. The N-terminal 12-residue peptide of Met-alpha(2)AP slows crosslinking of Met-alpha(2)AP(R6) or Met-alpha(2)AP(W6) by limiting access of factor XIIIa to Gln14 rather than shifting crosslinking to other Gln residues. Edman sequencing and mass analyses of tryptic peptides from each alpha(2)AP crosslinked with 5-(biotinamido)pentylamine showed Gln14 as the only major crosslinking site. Residues 5-8, GRQL in Met-alpha(2)AP(R6), and residues 1-8, MEPLGWQL in Met-alpha(2)AP(W6), slow fibrin crosslinking. CONCLUSION: Gln14 in both Met-alpha(2)AP(R6) and Met-alpha(2)AP(W6) is sheltered by the N-terminal 12-residue peptide, which, when cleaved, yields Asn-alpha(2)AP, which is rapidly crosslinked to fibrin and maximally protects it from plasmin. The R6 W polymorphism in Met-alpha(2)AP does not affect its crosslinking to fibrin, but it does slow cleavage by APCE and reduces the amount of Asn-alpha(2)AP available for rapid crosslinking to fibrin.

A randomized controlled trial comparing oxytocin administration before and after placental delivery in the prevention of postpartum hemorrhage.

OBJECTIVE: To determine if the timing of the administration of prophylactic oxytocin influences the incidence of postpartum hemorrhage caused by uterine atony, retained placenta, and third-stage duration. STUDY DESIGN: Parturients who presented for vaginal delivery were randomized in a double-blinded fashion to receive oxytocin, 20 units in a 500-mL crystalloid intravenous bolus, beginning upon delivery of either the fetal anterior shoulder or placenta. For all patients, the third stage of labor was managed with controlled cord traction until placental expulsion, followed by at least 15 seconds of fundal massage. Patients were excluded if they had a previous cesarean section, multiple gestation, antepartum hemorrhage, or bleeding disorder. RESULTS: A total of 1486 patients were enrolled: 745 in the before-placenta group and 741 in the after-placenta group. The groups were similar with respect to gestational age, fetal weight, labor duration, maternal age, parity, and ethnicity. The incidence of postpartum hemorrhage did not differ significantly between the two groups (5.4% vs 5.8%; crude OR, 0.92; 95% CI, 0.59 to 1.43). There were no significant differences between the two groups with respect to incidence of retained placenta (2.4% vs 1.6%; OR, 1.49; 95% CI, 0.72 to 3.08), or third-stage duration (7.7 minutes vs 8.1 minutes; P =.23). CONCLUSIONS: The administration of prophylactic oxytocin before placental delivery does not reduce the incidence of postpartum hemorrhage or third-stage duration, when compared with giving oxytocin after placental delivery. Early administration, however, does not increase the incidence of retained placenta.

Crosslinking of alpha 2-antiplasmin to fibrin.

Human alpha 2-antiplasmin (alpha 2AP) is the primary inhibitor of plasmin-mediated fibrinolysis and is an efficient substrate of activated factor XIII (FXIIIa). Among 452 amino acid residues in alpha 2AP, Gln2 is believed to be the sole FXIIIa-reactive site that participates in crosslinking alpha 2AP to fibrin. We studied the effect of mutating Gln2 on the ability of FXIIIa to catalyze crosslinking of alpha 2AP to fibrin. By FXIIIa catalysis, [14C]methylamine was incorporated into a Q2A-alpha 2AP mutant in which Gln2 (Q) was replaced by Ala (A), thereby indicating that wildtype alpha 2AP has more than one FXIIIa-reactive site. To identify the FXIIIa-reactive sites in alpha 2AP, wildtype alpha 2AP and Q2A-alpha 2AP were labeled with 5-(biotinamido)pentylamine by FXIIIa. Each labeled alpha 2AP was digested with trypsin and applied to an avidin affinity column to capture labeled peptides. Edman sequencing and mass analysis of each labeled peptide showed that out of 35 Gln residues in wildtype alpha 2AP, four were labeled with the following order of efficiency: Gln2 > Gln21 > Gln419 > Gln447. Q2A-alpha 2AP was also labeled at the three minor sites, Gln21 > Gln419 > Gln447. Q2A-alpha 2AP became crosslinked to fibirin(ogen) by FXIIIa catalysis at approximately one-tenth the rate of wt-alpha 2AP. These results demonstrate that alpha 2AP has one primary (Gln2) and three minor substrate sites for FXIIIa and that the three minor sites identified in this study can also participate in crosslink formation between alpha 2AP and fibrin, but at a much lower efficiency than the Gln2 site.

Cross-linking of wild-type and mutant alpha 2-antiplasmins to fibrin by activated factor XIII and by a tissue transglutaminase.

Human alpha(2)-antiplasmin (alpha(2)AP), the main inhibitor of plasmin-mediated fibrinolysis, is a substrate for plasma transglutaminase, also termed activated factor XIII (FXIIIa). Of 452 amino acids in alpha(2)AP, only Gln(2) is believed to be a fibrin-cross-linking (or FXIIIa-reactive) site. Kinetic efficiencies (k(cat)/K(m)((app))) of FXIIIa and the guinea pig liver tissue transglutaminase (tTG) and reactivities of Gln substrate sites were compared for recombinant wild-type alpha(2)AP (WT-alpha(2)AP) and Q2A mutant alpha(2)AP (Q2A-alpha(2)AP). [(14)C]Methylamine incorporation showed the k(cat)/K(m)((app)) of FXIIIa to be 3-fold greater than that of tTG for WT-alpha(2)AP. With FXIIIa or tTG catalysis, [(14)C]methylamine was incorporated into Q2A-alpha(2)AP, indicating that WT-alpha(2)AP has more than one Gln cross-linking site. To identify transglutaminase-reactive sites in WT-alpha(2)AP or Q2A-alpha(2)AP, each was labeled with 5-(biotinamido)pentylamine by FXIIIa or tTG catalysis. After each labeled alpha(2)AP was digested by trypsin, sequence and mass analyses of each labeled peptide showed that 4 of 35 Gln residues were labeled with the following reactivities: Gln(2) > Gln(21) > Gln(419) > Gln(447). Q(2)A-alpha(2)AP was also labeled at Gln(21) > Gln(419) > Gln(447), but became cross-linked to fibrin by FXIIIa or tTG at approximately one-tenth the rate for WT-alpha(2)AP. These results show that alpha(2)AP is a better substrate for FXIIIa than for this particular tTG, but that either enzyme involves the same Gln substrate sites in alpha(2)AP and yields the same order of reactivities.

Alpha-2 domain polymorphism and HLA class I peptide loading.

Diversity within the class I HLA antigen binding groove is positioned to moderate the presentation of peptide ligands. Polymorphism is widely dispersed about the peptide binding groove, and unravelling the functional significance of a given polymorphism requires comparative analysis of peptides presented by class I subtypes differing at the position(s) in question. Previous studies have demonstrated that not all class I polymorphisms act equally, and to determine the impact of substitutions specifically located in the alpha2 domain, peptides purified from B*1501, B*1512, B*1510, and B*1518 were examined by pooled Edman sequencing and comparative mass spectrometric analysis. Molecule B*1512 differs from B*1501 at residues 166 (Glu to Asp) and 167 (Trp to Gly) of the alpha2 domain. The pooled motif and ion mass ligand maps for B*1512 tightly matched those of B*1501, demonstrating that the 166/167 polymorphism between B*1501 and B*1512 has little impact upon ligand presentation. Although the 166/167 polymorphism minimally affects peptide binding preferences, this polymorphism makes B*1512 and B*1501 quite distinct by serology. We then compared the B70 molecules B*1510 and B*1518. The two are almost indistinguishable by serology and differ only by an alpha2 polymorphism at 116. Comparative peptide mapping shows that a Tyr to Ser polymorphism at 116 drastically changes the ligands bound by B*1510 and B*1518; no overlaps could be found. Polymorphisms in alpha2 therefore vary from subtle to extreme in the manner by which they moderate ligand presentation, and serologic crossreactivity did not reflect the ligands presented by these B15 subtypes.

Initiating ocular proteomics for cataloging bovine retinal proteins: microanalytical techniques permit the identification of proteins derived from a novel photoreceptor preparation.

Though some mechanisms of photoreception have been well characterized, others remain obscure. Presumably, most, if not all, of the major players in photoreceptor-specific functions are present in large amounts in the photoreceptor layer, and a catalog of these proteins will prove a useful tool for vision researchers. As a first step toward a complete catalog of photoreceptor cells, we have developed a novel method for isolating the photoreceptor cell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor layer had been removed, indicate that the preparation contains the photoreceptor outer segments and the majority of the inner segments. Proteins were extracted from the isolated photoreceptor cell layer as well as the rest of the retina with isoelectric focusing lysis buffer, and the protein components were separated by two-dimensional gel electrophoresis. The obtained protein maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested with trypsin, and the digests were extracted with solvent. The mixture of peptides digested from each protein was analyzed by high performance liquid chromatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Some of the peptides were isolated and their sequences were determined by gas phase Edman degradation. RNA transcripts extracted from the photoreceptor layer or the whole retina were subjected to Northern blot analysis as well as to reverse transcriptase-polymerase chain reaction amplification of probes for the successful selection of cDNA clones. These data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate aminotransferase and creatine kinase from the populations of abundant photoreceptor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photoreceptor functions by producing glutamate and ATP, respectively. We also identified two photoreceptor layer specific proteins: an acidic and high molecular weight protein, interphotoreceptor retinoid-binding protein, and an acidic and small molecular weight protein, recoverin.The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological condition. With the use of this technology, we anticipate that a researcher who knows only the 2-D gel position of a protein of interest can identify the protein, isolate a cDNA clone, and move into molecular genetic studies. Moreover, this streamlined technology will enable one to assemble a catalog of photoreceptor proteins using a minute amount of materials in a short period of time. We believe that such a catalog will serve as a valuable resource for vision investigators and will accelerate the rate of research progress.

Characterization of wild-type and mutant alpha2-antiplasmins: fibrinolysis enhancement by reactive site mutant.

During human blood clotting, alpha2-antiplasmin (alpha2AP) becomes covalently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in alpha2AP and a specific epsilon-lysyl group in each of the alpha-chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. We found that chemically Arg-modified alpha2AP, which lacked plasmin-inhibitory activity, competed effectively with native alpha2AP for becoming cross-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombinant alpha2AP reported to date by other groups either lacked or possessed a low level of FXIIIa substrate activity. As a first step in the development of an engineered protein that might have potential as a localized fibrin-specific fibrinolytic enhancer, we expressed recombinant alpha2AP in Pichia pastoris yeast. Two forms of nonglycosylated recombinant alpha2AP were expressed, isolated and characterized: (1) wild-type, which was analogous to native alpha2AP, and (2) a mutant form, which had Ala substituted for the reactive-site Arg364. Both the wild-type and mutant forms of alpha2AP functioned as FXIIIa substrates with affinities and kinetic efficiencies comparable to those of native alpha2AP, despite each having an additional acetylated Met blocking group at their respective amino-termini. Wild-type recombinant alpha2AP displayed full plasmin inhibitory activity, while mutant alpha2AP had none. Neither the absence of glycosylation nor blockage of the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activities of wild-type alpha2AP. When our mutant alpha2AP, which lacked plasmin-inhibitory function, was added to human plasma or whole blood clots, urokinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, indicating that mutant alpha2AP augmented lysis by competing with native alpha2AP for FXIIIa-catalyzed incorporation into fibrin.

HLA-B15 peptide ligands are preferentially anchored at their C termini.

Therapies to elicit protective CTL require the selection of pathogen- and tumor-derived peptide ligands for presentation by MHC class I molecules. Edman sequencing of class I peptide pools generates "motifs" that indicate that nonameric ligands bearing conserved position 2 (P2) and P9 anchors provide the optimal search parameters for selecting immunogenic epitopes. To determine how well a motif represents its individual constituents, we used a hollow-fiber peptide production scheme followed by the mapping of endogenously processed class I peptide ligands through reverse-phase HPLC and mass spectrometry. Systematically mapping and characterizing ligands from B*1508, B*1501, B*1503, and B*1510 demonstrate that the peptides bound by these B15 allotypes i) vary in length from 7 to 12 residues, and ii) are more conserved at their C termini than their N-proximal P2 anchors. Comparative peptide mapping of these B15 allotypes further pinpoints endogenously processed ligands that bind to the allotypes B*1508, B*1501, and B*1503, but not B*1510. Overlapping peptide ligands are successful in binding to B*1501, B*1503, and B*1508 because these B15 allotypes share identical C-terminal anchoring pockets whereas B*1510 is divergent in the C-terminal pocket. Therefore, endogenous peptide loading into the B15 allotypes requires that a conserved C terminus be anchored in the appropriate specificity pocket while N-proximal anchors are more flexible in their location and sequence. Queries for overlapping and allele-specific peptide ligands may thus be contingent on a conserved C-terminal anchor.

Effect of phenylglyoxal-modified alpha2-antiplasmin on urokinase-induced fibrinolysis.

One of the functions of activated blood clotting factor XIII (FXIIIa) is the crosslinking of alpha2-antiplasmin (alpha2AP) to fibrin. This process results in localization and concentration of alpha2AP throughout fibrin, thereby making fibrin more resistant to digestion by plasmin. We reasoned that competition by chemically-modified inactive alpha2AP (mod alpha2AP) with native alpha2AP would diminish the resistance of fibrin to digestion by plasmin. Mod alpha2AP was prepared by treating native alpha2AP with an Arg-specific reagent, phenylglyoxal. An average of four of the total nineteen Arg residues in alpha2AP reacted with phenylglyoxal and resulted in complete loss of plasmin inhibitory activity; however, mod alpha2AP competed effectively with native alpha2AP for becoming crosslinked to fibrin by FXIIIa catalysis. In the presence of mod alpha2AP, urokinase (UK)-induced plasma clot lysis time shortened significantly. Mod alpha2AP enhanced UK-induced clot lysis in a whole blood system as shown by the similarities of rates of clot lysis for a mixture of 20 U/ml UK and 1.5 microM mod alpha2AP versus that induced by 100 U/ml UK without mod alpha2AP. Less fibrinogenolysis occurred in whole blood when mod alpha2AP was present since much lower UK concentrations were needed to achieve the same level of fibrinolysis than when only native alpha2AP was present. Our results indicate that mod alpha2AP enhances UK-induced fibrinolysis by competitive inhibition of factor XIIIa-mediated incorporation of native alpha2AP into fibrin.

Complexity among constituents of the HLA-B*1501 peptide motif.

Analysis of peptides derived from HLA class I molecules indicates that thousands of unique peptides are bound by a single molecular type, and sequence examination of the pooled constituents yields a motif which collectively defines the peptides bound by a given class I molecule. Motifs resulting from pooled sequencing are then used to infer whether particular viral and tumor protein fragments might serve as class I-presented peptide therapeutics. Still undetermined from a pooled motif is the breadth or range of peptides in the population which are brought together to form the pooled motif, and it is therefore not yet known how representative of the population a pooled motif is. By employing hollow fiber bioreactors for large-scale production of HLA class I molecules, sufficient peptides are produced to investigate individual subsets of peptides comprising a motif. Edman sequencing and mass spectrometric analysis of peptides eluted from HLA-B*1501 reveal that many peptide sequences fail to align with either the N- or C-terminal anchors predicted for the B*1501 peptide motif through whole pool sequencing. These analyses further reveal auxiliary anchors not previously detected and peptides significantly larger and smaller than the predicted nonamer, ranging from 6 to 12 amino acids in length. These results demonstrate that constituents of the B*1501 peptide pool vary markedly in comparison with one another and therefore in comparison with previously established B*1501 motifs, and such complexity indicates that many of the peptide ligands presented to CTL cannot be predicted using class I consensus motifs as search criteria.

Mass spectrometric characterization and glycosylation profile of bovine pancreatic bile salt-activated lipase.

We developed a procedure for the large scale isolation of bovine bile salt-activated lipase (BAL) for its crystallization [Wang, X., et al. (1997) Structure 5, 1209-1218] and also carried out a study on the molecule's glycosylation profile for a better deduction of the structure of the enzyme. Mass spectrometric analysis of the CNBr-generated peptides indicated that only one (Asn-361) of the two potential N-glycosylation sites (Asn-187 and Asn-361) with NXT motif is glycosylated. The analysis of the isolated CNBr peptide containing Asn-361 showed that it existed in three glycoforms in a ratio of 1.0:2.8:1.0, with oligosaccharide moieties weighing 1900.1, 2045.2, and 2336.4 Da, respectively. The major oligosaccharide chain contained mannose: galactose:N-acetylglucosamine:fucose:sialic acid in a molar ratio of 2:2:4:2:1. It was also determined that the potential O-glycosylated peptide (CB13) is not O-glycosylated and, in addition, it was found that there was microheterogeneity in the C-terminus of the isolated bovine BAL. The results obtained from this mass spectrometric study combined with the X-ray crystallographic studies provide more precise structural information on BAL. The procedure described here for the mass spectrometric analysis of CNBr-generated peptides also has general applicability for analysis of the glycosylation profile of glycoproteins and the C-terminal peptide structure of proteins.

Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli.

A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step.

Purification of human alpha 2-antiplasmin with chicken IgY specific to its carboxy-terminal peptide.

alpha 2-antiplasmin, a plasma glycoprotein of the serpin superfamily, is the primary physiological inhibitor of plasmin, the key enzyme in fibrin degradation. Previous purification methods utilize lengthy multistep protocols with low yields or use monoclonal antibodies that are expensive or difficult to make. With a relatively small investment, a chicken was immunized with keyhole limpet hemocyanin-conjugated to alpha 2-antiplasmin C-terminal 26 residue synthetic peptide and the peptide-specific antibody (IgY) was isolated from the egg yolks of hens using the peptide affinity column. Based on the interaction between this IgY and alpha 2-antiplasmin, pure alpha 2-antiplasmin was isolated from human plasma in two steps: (a) citrated plasma was precipitated with 15% PEG-8000 to remove the bulk of plasma proteins while retaining the majority of alpha 2-antiplasmin activity; and (b) the alpha 2-antiplasmin was affinity-purified from the supernatant using the IgY column. Yields were typically 48% and the purity and authenticity of the alpha 2-antiplasmin were verified by gel electrophoresis, Western Blot analysis, N-terminal sequence, and amino acid analysis.

Residues within the polycationic region of cGMP phosphodiesterase gamma subunit crucial for the interaction with transducin alpha subunit. Identification by endogenous ADP-ribosylation and site-directed mutagenesis.

Interaction between the gamma subunit (Pgamma) of cGMP phosphodiesterase and the alpha subunit (Talpha) of transducin is a key step for the regulation of cGMP phosphodiesterase in retinal rod outer segments. Here we have utilized a combination of specific modification by an endogenous enzyme and site-directed mutagenesis of the Pgamma polycationic region to identify residues required for the interaction with Talpha. Pgamma, free or complexed with the alphabeta subunit (Palphabeta) of cGMP phosphodiesterase, was specifically radiolabeled by prewashed rod membranes in the presence of [adenylate-32P]NAD. Identification of ADP-ribose in the radiolabeled Pgamma and radiolabeling of arginine-replaced mutant forms of Pgamma indicate that both arginine 33 and arginine 36 are similarly ADP-ribosylated by endogenous ADP-ribosyltransferase, but only one arginine is modified at a time. Pgamma complexed with Talpha (both GTP- and GDP-bound forms) was not ADP-ribosylated; however, agmatine, which cannot interact with Talpha, was ADP-ribosylated in the presence of Talpha, suggesting that a Pgamma domain containing these arginines is masked by Talpha. A Pgamma mutant (R33,36K), as well as wild type Pgamma, inhibited both GTP hydrolysis of Talpha and GTP binding to Talpha. Moreover, GTP-bound Talpha activated Palphabeta that had been inhibited by R33,36K. However, another Pgamma mutant (R33,36L) could not inhibit these Talpha functions. In addition, GTP-bound Talpha could not activate Palphabeta inhibited by R33,36L. These results indicate that a Pgamma domain containing these arginines is required for its interaction with Talpha, but not with Palphabeta, and that positive charges in these arginines are crucial for the interaction.

Large-scale production of class I bound peptides: assigning a signature to HLA-B*1501.

A peptide-based vaccine must be bound and presented by major histocompatibility complex class I molecules to elicit a CD8(+) T-cell response. Because class I HLA molecules are highly polymorphic, it has yet to be established how well a vaccine peptide that stimulates one individual's CD8(+) cytotoxic T lymphocytes will be presented by a second individual's different class I molecules. Therefore, to facilitate precise comparisons of class I peptide binding overlaps, we uniquely combined hollow-fiber bioreactors and mass spectrometry to assign precise peptide binding signatures to individual class I HLA molecules. In applying this strategy to HLA-B*1501, we isolated milligram quantities of B*1501-bound peptides and mapped them using mass spectrometry. Repeated analyses consistently assign the same peptide binding signature to B*1501; the degree of peptide binding overlap between any two class I molecules can thus be determined through comparison of their peptide signatures.

Peer and community leader education to prevent youth violence.

The program described here tests the effectiveness of a community-based and school-based program to reduce violence among African-American and Hispanic adolescents. The program methods are based on social network theory research, which has found that key lay people in communities can be identified and trained to carry out prevention programs. The educational content is based on theories suggesting that characteristics of healthy, adaptive individuals and communities can be taught. A violence-prevention leadership program is provided to a cohort of middle-school student peer leaders and their parents and for the leaders of the neighborhoods around the middle schools. Three matched pairs of urban middle-school attendance zones were randomly assigned to receive either the intervention or serve as a nonintervention control group. Surveys, interviews, and observations were conducted with the peer leaders, their parents, community leaders, and community residents. Sixty-six percent of the peer leaders reported that they had hit someone in the past 30 days. Twenty-six percent of the sixth-graders had punched or beaten someone in the past 30 days. Within the past year, 6% of the adults had slapped or kicked someone. Within the past 30 days, 14% of the sixth-graders had been punched or beaten. Within the past year, 6% of the adults had been punched or beaten. A large percentage of adolescents are victims and perpetrators of violence and are exposed to violence in their neighborhoods. Violence-prevention strategies can be implemented through collaborations among health departments, community-based organizations, universities, and schools.

Isolation and characterization of recombinant human apolipoprotein C-II expressed in Escherichia coli.

A full-length recombinant human apolipoprotein C-II (ApoC-II) has been successfully expressed in Escherichia coli using the T7 expression system. The recombinant ApoC-II. which was expressed intracellularly in the inclusion bodies, was solubilized with 8 M urea and purified using Sephadex G-75 gel permeation chromatography. Four liters of the bacterial culture yielded 16-20 mg of purified recombinant ApoC-II. Sequencing and mass spectrometric analyses indicated that the isolated recombinant ApoC-II contained predominantly (64%) the native form with threonine as the N-terminus, but also contained a minor (36%) molecular form of ApoC-II with an additional methionine at the N-terminus (Met-ApoC-II). Analysis of the recombinant ApoC-II by tryptic digestion and high performance liquid chromatography-electrospray mass spectrometry provides additional conclusive evidence that, with the exception of the N-terminus of Met-ApoC-II, the expressed ApoC-II has the expected peptide sequence. However, this extra N-terminal methionine residue can be excised by further in vitro treatment with methionine aminopeptidase. The purified recombinant ApoC-II was found to be competent in the activation of bovine milk lipoprotein lipase. Thus, the recombinant ApoC-II prepared from E. coli may have a pharmacological application for the treatment of patients with genetic hypertriglyceridemia caused by ApoC-II deficiency.

An RNA polymerase II elongation factor encoded by the human ELL gene.

The human ELL gene on chromosome 19 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemias. Here, ELL was shown to encode a previously uncharacterized elongation factor that can increase the catalytic rate of RNA polymerase II transcription by suppressing transient pausing by polymerase at multiple sites along the DNA. Functionally, ELL resembles Elongin (SIII), a transcription elongation factor regulated by the product of the von Hippel-Lindau (VHL) tumor suppressor gene. The discovery of a second elongation factor implicated in oncogenesis provides further support for a close connection between the regulation of transcription elongation and cell growth.