Ex vivo analysis of T-cell responses to Epstein-Barr virus-encoded oncogene latent membrane protein 1 reveals highly conserved epitope sequences in virus isolates from diverse geographic regions

Epstein-Barr virus (EBV)-encoded oncogene latent membrane protein (LMP) 1, which is consistently expressed in multiple EBV-associated malignancies, has been proposed as a potential target antigen for any future vaccine designed to control these malignancies. However, the high degree of genetic variation in the LMP1 sequence has been considered a major impediment for its use as a potential immunotherapeutic target for the treatment of EBV-associated malignancies. In the present study, we have employed a highly efficient strategy, based on ex vivo functional assays, to conduct an extensive sequence-wide analysis of LMP1-specific T-cell responses in a large panel of healthy virus carriers of diverse ethnic origin and nasopharyngeal carcinoma patients. By comparing the frequencies of T cells specific for overlapping peptides spanning LMP1, we mapped a number of novel HLA class I- and class II-restricted LMP1 T-cell epitopes, including an epitope with dual HLA class I restriction. More importantly, extensive sequence analysis of LMP1 revealed that the majority of the T-cell epitopes were highly conserved in EBV isolates from Caucasian, Papua New Guinean, African, and Southeast Asian populations, while unique geographically constrained genetic variation was observed within one HLA A2 supertype-restricted epitope. These findings indicate that conserved LMP1 epitopes should be considered in designing epitope-based immunotherapeutic strategies against EBV-associated malignancies in different ethnic populations.

The effect of protein binding on the hepatic first pass of O-acyl salicylate derivatives in the rat

In this work the in-situ perfused rat liver has been used to examine the effect of changing the protein content of the perfusate on the hepatic extraction of O-acyl esters of salicylic acid. The hepatic availability (F) of these solutes was studied at a flow-rate of 30 mt min(-1) with perfusate albumin concentrations of 0, 2, and 4% w/v. The hepatic availability of the esters was shown to decrease with increasing carbon-chain length in the O-acyl group; for all the esters the hepatic availability increased with increasing albumin concentration in the perfusate. The dispersion-model-derived efficiency number (R-N) Of the esters was shown to increase with increasing lipophilicity and decrease with increasing albumin concentration in the perfusate. The unbound fraction (f(u),) of the esters decreased with lipophilicity. R-N/f(u), for acetylsalicylic acid remained relatively constant as the albumin concentration was increased. However, R-N/f(u), for n-pentanoyl- and n-hexanoylsalicylic acids increased significantly as albumin concentration increased from 0% to 4%. Thus, for the more lipophilic solutes (n-pentanoyl- and n-hexanoylsalicylic acids) the presence of albumin apparently facilitates the uptake of unbound solute relative to acetylsalicylic acid.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Derivatives of 1,3,5-triamino-1,3,5-trideoxy-cis-inositas versatile pentadentate ligands for protein labeling with Re-186/188. Prelabeling, biodistribution, and X-ray structural studies

The pentadentate H(3)bhci [1,3,5-trideoxy-1,3-bis((2-hydroxybenzyl)amino)-cis-inistol] and its bifunctionalized analogue H(3)bhci-glu-H [1,3,5-trideoxy-1,3-bis((2-hydroxybenzyl)amino)-5-glutaramido-cis-inositol] were synthesized, and their coordination chemistry was investigated with inactive rhenium, with no carrier added Re-188 and with carrier added Re-186. The neutral Re(V) complexes [ReO-(bhci)] and [ReO(bhci-glu-H)] are formed in good yields starting from [ReOCl3(P(C6H5)(3))(2)] or in quantitative yield directly from [(ReO4)-Re-186/188](-) in aqueous solution by reduction with Sn(II) or Sn(0). The X-ray structures of [ReO(bhci)] and [ReO(bhci-glu-H)] were elucidated revealing pentadentate side on coordination of the ligands to the Re=O core. The basic cyclohexane frame adopts a chair form in the case of [ReO(bhci)] and a twisted boat form in the case of [ReO(bhci-glu-H)]. [ReO(bhci)] crystallizes in the monoclinic space group C2/c with a = 27.425(3), b = 14.185(1), c = 19.047(2) Angstrom, and beta = 103.64(2)degrees and [ReO(bhci-glu-H)] in the monoclinic space group P2(1)/c with a = 13.056(3), b = 10.180(1), c = 22.378(5) Angstrom and beta = 98.205(9)degrees Both Re-188 complexes are stable in human serum for at least 3 days without decomposition. After injection into mice, [ReO(bhci-glu)](-) is readily excreted through the intestines, while [ReO(bhci)] is excreted by intestines, liver, and the kidneys. TLC investigations of the urine showed exclusively the complexes [ReO(bhci-glu-H)] and [ReO(bhci)], respectively, and no decomposition products. For derivatization of antibodies, the carboxylic group of [ReO(bhci-glu-H)] was activated with N-hydroxysuccinimide, which required unusually vigorous reaction conditions (heating). The anti colon cancer antibody mAb-35 [IgG and F(ab')(2) fragment] was labeled with [(ReO)-Re-186/188(bhci-glu)] to a specific activity of up to 1.5 mCi/mg (55 MBq/mg) with full retention of immunoreactivity. Labeling yields followed pseudo-first-order kinetics in antibody concentration with the ratio of rates between aminolysis and hydrolysis being about 2. Biodistributions of Re-186-labeled intact mAb-35 as well as of its F(ab')(2) fragment in tumor-bearing nude mice revealed good uptake by the tumor with only low accumulation of radioactivity in normal tissue.

Post translational modifications of recombinant human Papillomavirus type 6b major capsid protein

We have determined the post-translational modifications of the major capsid protein, L1 of human papillomavirus (HPV) type 6b. Since this virus cannot be cultured in the laboratory to obtain sufficient material for a study, a recombinant L1 protein produced in a vaccinia virus expression system was used in this investigation. Our results show that this protein is phosphorylated at serine residues and is also glycosylated. No myristoylation or palmitoylation was detected. The fraction of L1 protein incorporated into virus-like particles was not glycosylated. Since recombinant L1 protein is a potential human vaccine candidate, knowledge of the post-translation modifications of this protein may prove useful for the design of anti-HPV vaccines. (C) 1999 Elsevier Science B.V. All rights reserved.

Active site-directed protein regulation

Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effecters bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.

Characterization of the acyl carrier protein gene and the fab gene locus in Xanthomonas albilineans

A genomic region containing the fatty acid biosynthetic (fab) genes was isolated from the sugarcane leaf-scald pathogen Xanthomonasalbilineans. The order and predicted products of fabG (beta -ketoacyl reductase), acpP (acyl carrier protein), fabF(ketoacyl synthase II) and downstream genes in X. albilineans are very similar to those in Escherichia coli, with one exception. Sequence analysis, confirmed by insertional knockout and specific substrate feeding experiments, shows that the position occupied by pabC (encoding aminodeoxychorismate lyase) in other bacteria is occupied instead by pabB (encoding aminodeoxychorismate synthase component I) in X. albilineans. Downstream of pabB, X. albilineans resumes the arrangement common to characterized Gram-negative bacteria, with three transcriptionally coupled genes, encoding an ORF340 protein of undefined function, thymidylate kinase and delta' subunit of DNA polymerase III holoenzyme (HolB). Different species may obtain a common advantage from coordinated regulation of the same biosynthetic pathways using different genes in this region. (C) 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Effect of shear stress on expression of a recombinant protein by Chinese hamster ovary cells

A flow chamber was used to impart a steady laminar shear stress on a recombinant Chinese hamster ovary (CHO) cell line expressing human growth hormone (hGH). The cells were subjected to shear stress ranging from 0.005 to 0.80 N m(-2). The effect of shear stress on the cell specific glucose uptake, cell specific hGH, and lactate productivity rates were calculated. No morphological changes to the cells were observed over the range of shear stresses examined. When the cells were subjected to 0.10 N m(-2) shear in protein-free media without Pluronic F-68, recombinant protein production ceased with no change in cell morphology, whereas control cultures were expressing hGH at 0.35 mug/10(6) cells/h. Upon addition of the shear protectants, Pluronic F-68 (0.2% [w/v]) or fetal bovine serum (1.0% [v/v] FBS), the productivity of the cells was restored. The effect of increasing shear stress on the cells in protein-free medium containing Pluronic F-68 was also investigated. Cell specific metabolic rates were calculated for cells under shear stress and for no-shear control cultures performed in parallel, with shear stress rates expressed as a percentage of those obtained for control cultures. Upon increasing shear from 0.005 to 0.80 N m(-2), the cell specific hGH productivity decreased from 100% at 0.005 N m(-2) to 49% at 0.80 N m(-2) relative to the no-shear control. A concurrent increase in the glucose uptake rate from 115% at 0.01 N m(-2) to 142% at 0.80 N m(-2), and decreased lactate productivity from 92% to 50%, revealed a change in the yield of products from glucose compared with the static control. It was shown that shear stress, at sublytic levels in medium containing Pluronic F-68, could decrease hGH specific productivity. (C) 2002 Wiley Periodicals, Inc.

CysView: protein classification based on cysteine pairing patterns

CysView is a web-based application tool that identifies and classifies proteins according to their disulfide connectivity patterns. It accepts a dataset of annotated protein sequences in various formats and returns a graphical representation of cysteine pairing patterns. CysView displays cysteine patterns for those records in the data with disulfide annotations. It allows the viewing of records grouped by connectivity patterns. CysView's utility as an analysis tool was demonstrated by the rapid and correct classification of scorpion toxin entries from GenPept on the basis of their disulfide pairing patterns. It has proved useful for rapid detection of irrelevant and partial records, or those with incomplete annotations. CysView can be used to support distant homology between proteins. CysView is publicly available at http://research.i2r.a-star.edu.sg/CysView/.

Reorganization of terminator DNA upon binding replication terminator protein: Implications for the functional replication fork arrest complex

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B. subtilis DNA terminator, Terl, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of Terl causes the DNA to become slightly unwound and bent by similar to 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to similar to 60 degrees. We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner, in the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry at the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Purification by reflux electrophoresis of whey proteins and of a recombinant protein expressed in Dictyostelium discoideum

Protein purification that combines the use of molecular mass exclusion membranes with electrophoresis is particularly powerful as it uses properties inherent to both techniques. The use of membranes allows efficient processing and is easily scaled up, while electrophoresis permits high resolution separation under mild conditions. The Gradiflow apparatus combines these two technologies as it uses polyacrylamide membranes to influence electrokinetic separations. The reflux electrophoresis process consists of a series of cycles incorporating a forward phase and a reverse phase. The forward phase involves collection of a target protein that passes through a separation membrane before trailing proteins in the same solution. The forward phase is repeated following clearance of the membrane in the reverse phase by reversing the current. We have devised a strategy to establish optimal reflux separation parameters, where membranes are chosen for a particular operating range and protein transfer is monitored at different pH values. In addition, forward and reverse phase times are determined during this process. Two examples of the reflux method are described. In the first case, we describe the purification strategy for proteins from a complex mixture which contains proteins of higher electrophoretic mobility than the target protein. This is a two-step procedure, where first proteins of higher mobility than the target protein are removed from the solution by a series of reflux cycles, so that the target protein remains as the leading fraction. In the second step the target protein is collected, as it has become the leading fraction of the remaining proteins. In the second example we report the development of a reflux strategy which allowed a rapid one-step preparative purification of a recombinant protein, expressed in Dictyostelium discoideum. These strategies demonstrate that the Gradiflow is amenable to a wide range of applications, as the protein of interest is not necessarily required to be the leading fraction in solution. (C) 1997 Elsevier Science B.V.

Synthesis, identification, characterization, stability, solubility, and protein binding of ester derivatives of salicylic acid and diflunisal

O-Acyl esters were prepared from salicylic acid and diflunisal by esterification with the appropriate acyl anhydride (in the presence of sulfuric acid at 80 degrees C) or acyl chloride (in the presence of pyridine at 0 degrees C). Synthesis, identification and characterization of these compounds is described. In vitro hydrolysis, solubility and protein binding studies of these O-acyl esters were performed. For the diflunisal esters, the melting points fell as the side chain was increased from ethyl to pentyl. The melting points showed no significant difference as the length of the side chain was increased from pentyl to heptyl. The aspirin analogues showed a similar trend, The relationship between solubility and carbon chain length agreed closely with that for the melting points with carbon chain length. In vitro non-enzymatic hydrolysis studies concluded that: (1) hydrolysis rate constants generally decreased with carbon chain length; (2) the diflunisal esters have shorter half lives compared with their salicylate counterparts; and (3) the in vitro hydrolysis of these compounds was retarded by the presence of bovine serum albumin. Protein binding experiments showed that the strength of binding of the aspirin and diflunisal analogues to bovine serum albumin increased with carbon chain length. (C) 1997 Elsevier Science B.V.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

Down-regulation of the amyloid protein precursor of Alzheimer's disease by antisense oligonucleotides reduces neuronal adhesion to specific substrata

The hallmark of Alzheimer's disease is the cerebral deposition of amyloid which is derived from the amyloid precursor protein (APP). The function of APP is unknown but there is increasing evidence for the role of APP in cell-cell and/or cell-matrix interactions. Primary cultures of murine neurons were treated with antisense oligonucleotides to down-regulate APP. This paper presents evidence that APP mediates a substrate-specific interaction between neurons and extracellular matrix components collagen type I, laminin and heparan sulphate proteoglycan but not fibronectin or poly-L-lysine. It remains to be determined whether this effect is the direct result of APP-matrix interactions, or whether an intermediary pathway is involved. (C) 1997 Elsevier Science B.V.