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J Shiloach

Publications and source records attributed to J Shiloach.

At least 37 records · Page 2Linked to original sources
PubMed1998

Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation.

The productivity of Escherichia coli as a producer of recombinant proteins is affected by its metabolic properties, especially by acetate production. Two commercially used E. coli strains, BL21 (lambdaDE3) and JM109, differ significantly in their acetate production during batch fermentation at high initial glucose concentrations. E. coli BL21 grows to an optical density (OD, 600 nm) of 100 and produces no more than 2 g/L acetate, while E. coli JM109 grows to an OD (600 nm) of 80 and produces up to 14 g/L acetate. Even in fed-batch fermentation, when glucose concentration is maintained between 0.5 and 1.0 g/L, JM109 accumulates 4 times more acetate than BL21. To investigate the difference between the two strains, metabolites and enzymes involved in carbon utilization and acetate production were analyzed (isocitrate, ATP, phosphoenolpyruvate, pyruvate, isocitrate lyase, and isocitrate dehydrogenase). The results showed that during batch fermentation isocitrate lyase activity and isocitrate concentration were higher in BL21 than in JM109, while pyruvate concentration was higher in JM109. The activation of the glyoxylate shunt pathway at high glucose concentrations is suggested as a possible explanation for the lower acetate accumulation in E. coli BL21. Metabolic flux analysis of the batch cultures supports the activity of the glyoxylate shunt in E. coli BL21.

Acetic Acid↗
PubMed1997

Treatment of uveitis by oral administration of retinal antigens: results of a phase I/II randomized masked trial.

PURPOSE: To evaluate the effect and safety of the oral administration of retinal antigens as a treatment of ocular inflammation. METHODS: In a phase I/II randomized masked trial, patients with endogenous uveitis who were dependent on immunosuppressive agents were randomly assigned to receive either retinal S antigen alone (10 patients), retinal S antigen and a mixture of soluble retinal antigens (10 patients), a mixture of soluble retinal antigens alone (10 patients), or placebo (15 patients). An attempt was then made to taper patients completely off their standard immunosuppressive therapy over an 8 week period. The primary study endpoint was time to ocular inflammatory attack. The secondary study endpoint was the ability to taper patients completely off their immunosuppressive or cytotoxic medication within 8 weeks. RESULTS: Time to development of the main study endpoint was not statistically significantly different for any of the four treatment groups. However, the group receiving the purified S antigen alone appeared to be tapered off their immunosuppressive medication more successfully compared with patients given placebo (P = .08), whereas all the other groups appeared to do worse than did those receiving placebo. No toxic effects attributable to any treatment were observed. CONCLUSIONS: This phase I/II study is the first to test the use of orally administered S antigen in the treatment of uveitis. Although not statistically significant, patients given S antigen were more likely to be tapered off their chronically administered systemic immunosuppressive therapy than were the other groups tested.

Administration, Oral↗
PubMed1997

Correction of the NMR structure of the ETS1/DNA complex.

The ETS family of transcription factors consists of a group of proteins that share a highly conserved 85 amino acid DNA-binding domain (DBD). This family recognizes a consensus sequence rich in purine bases with a central GGAA motif. A comparison of the published three-dimensional structures of the DBD/DNA complexes of ETS1 by NMR [Werner et al. (1995) Cell, 83, 761-771] and the related Pu.1 by X-ray crystallography [Kodandapani et al. (1996) Nature, 380, 456-460] reveals an apparent discrepancy in which the protein domains bind with opposite polarity to their target sequences. This surprising and highly unlikely result prompted us to reexamine our NMR structure. Additional NMR experiments now reveal an error in the original interpretation of the spectra defining the orientation of the ETS1-DBD on DNA. It was originally reported that the ETS1-DBD bound to DNA with a bipartite motif involving major groove recognition via a helix-turn-helix element and minor groove recognition via protein side-chain intercalation. The presence of intercalation was deduced on the basis of numerous NOEs between several amino acids in the protein and a resonance at 12.33 ppm originally assigned to a DNA imino proton. New NMR experiments now conclusively demonstrate that this resonance, which is located within the DNA imino proton region of the spectrum, arises from the hydroxyl proton of Tyr86. Realization of this error necessitated reanalysis of the intermolecular NOEs. This revealed that the orientation of the ETS1-DBD in the complex is opposite to that originally reported and that a tryptophan residue does not intercalate into the DNA. The calculation of a new ensemble of structures based on the corrected data indicates that the structure of the ETS1-DBD/DNA complex is indeed similar to the X-ray structure of the Pu.1-DBD/DNA complex.

Amino Acid Sequence↗
PubMed1997

Permissive recognition of immunodominant determinants of the retinal S-antigen in different rat strains, primates and humans.

The majority of antigenic peptides exhibit restriction in their interaction with the MHC molecules on antigen-presenting cells of different haplotypes. Certain peptides, however, are "permissive': they bind strongly to different MHC molecules and are selected as the immunodominant epitopes by animals using these MHC gene products. Here we show for the first time that several peptides from four regions of the sequence of human S-antigen (H-SAg), a retinal-specific protein, demonstrate high levels of permissiveness. Each of these peptides was found to be immunodominant in at least some of four inbred rat strains and five cynomolgus monkeys, immunized with whole H-SAg. Moreover, some of these peptides were recognized by lymphocytes from four normal controls and four patients with uveitis who responded against the H-SAg molecule. On the other hand, the permissive peptides stimulated marginal or no response in cultures of Lewis rats injected with adjuvant alone, or rat and human cell lines specific to other antigens, thus demonstrating that these peptides do not carry any non-specific mitogenic activity. One peptide, 29, which was found immunodominant in the monkeys, the uveitis patients and Lewis rats, is highly immunopathogenic in this rat strain. No good correlation between immunodominance and immunopathogenicity was found with other H-SAg peptides. The finding of cross-species permissiveness among peptides of H-SAg and similar observations with myelin proteins suggest that permissiveness could be quite prevalent among peptides of immunopathogenic antigens.

Adult↗
PubMed1997

Synthesis and immunological properties of Vi and di-O-acetyl pectin protein conjugates with adipic acid dihydrazide as the linker.

The Vi capsular polysaccharide of Salmonella typhi, a licensed vaccine for typhoid fever in individuals > or = 5 years old, induces low and short-lived antibodies in children, and reinjection does not elicit booster responses at any age. Its immunogenicity was improved by binding Vi to proteins by using N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as a linker. Similar findings were observed with the structurally related, di-O-acetyl derivative of pectin [poly-alpha(1-->4)-D-GalpA] designated OAcP. Protein conjugates of Vi and OAcP were synthesized by carbodiimide-mediated synthesis with adipic acid dihydrazide (ADH) as the linker. Hydrazide groups were introduced into proteins (bovine serum albumin or recombinant Pseudomonas aeruginosa exoprotein A) by treatment with ADH and 1-ethyl-3(3-dimethylaminopropyl carbodiimide (EDC). The resultant adipic acid hydrazide derivatives (AH-proteins), containing 2.3 to 3.4% AH, had antigenic and physicochemical properties similar to those of the native proteins. The AH-proteins were bound to Vi and OAcP by treatment with EDC. The immunogenicity of Vi or OAcP, alone or as protein conjugates, was evaluated in young outbred mice and guinea pigs by subcutaneous injection of 2.5 and 5.0 microg, respectively, of polysaccharide, and antibodies were measured by enzyme-linked immunosorbent assay. All conjugates were significantly more immunogenic than Vi or OAcP alone and induced booster responses with 5- to 25-fold increases of antibodies. Vi conjugates were significantly more immunogenic than their OAcP analogs. A carboxymethyl derivative of yeast beta-glucan enhanced the anti-Vi response elicited by an OAcP conjugate but had no effect on the immunogenicity of Vi or of OAcP alone. Vi and OAcP conjugates synthesized by this scheme will be evaluated clinically.

Adipates↗
PubMed1996

Large scale recovery and purification of periplasmic recombinant protein from E. coli using expanded bed adsorption chromatography followed by new ion exchange media.

Expanded bed chromatography was used for the recovery and purification of modified Pseudomonas aeruginosa exotoxin A. The exotoxin accumulates in the periplasmic space of E. coli BL21 (lambda DE3), was released from the cells by osmotic shock and captured by applying the open cell suspension directly to an anion exchanger (STREAMLINE DEAE) using an expanded bed (STREAMLINE) column. Processing of 4.5 kg of E. coli using the expanded bed process was 3 times faster and did not require clarification of the bacterial extract, in comparison with the conventional purification method. Also, the recovered protein solution was 3 times more concentrated and the yield slightly higher.

ADP Ribose Transferases↗
PubMed1996

Human lymphocyte responses against epitopes of a self antigen: a follow-up at different time points.

This study was aimed at examining the changes that may occur with time in the lymphocyte responses of an individual against peptide determinants of self antigens. Peripheral blood lymphocytes were collected from the same donor at seven different time points during a 10-month period and tested for their proliferative responses against whole human S-antigen and 40 overlapping peptides derived from the sequence of this uveitogenic retinal protein. Lymphocytes collected at different dates varied in their responsiveness to all tested antigens, as well as in the pattern of their response against individual peptides. The latter variability was expressed by differences in the hierarchical order of the most stimulatory peptides for lymphocytes collected at the seven time points. Despite the variability in their stimulatory capacity, six of the peptides exhibited their immunodominance by eliciting proliferation in lymphocytes collected at all time points. Our results thus indicate that data collected from a single sample of blood may be insufficient to accurately assess the level of cellular response to autologous antigens.

Amino Acid Sequence↗
PubMed1996

A new reagent for the induction of T-cell depletion, anti-CD3-CRM9.

We have developed a new reagent for inducing in vivo T-cell depletion and have tested this reagent in rhesus monkeys. The reagent is an anti-CD3 epsilon immunotoxin based on a diphtheria toxin binding-site mutant, CRM9. After administration to monkeys, T cells are depleted from both the blood and lymph node compartments to < 1% of their initial values. T-cell depletion is associated with transient immunosuppression, as judged by delayed rejection of RhLA-mismatched skin allografts. T cells are repopulated in both compartments; however, the rate of repopulation is age dependent. The rate is rapid in juvenile animals (12 days) and requires > 30 days in old animals. The correlation between repopulation rate and age suggests that the repopulation is thymus dependent and that the repopulated T cells are probably naive T cells. This reagent should be a valuable tool in studying the role of memory T cells in rhesus models of autoimmune diseases and protocols of tolerance induction after organ transplantation.

Animals↗
PubMed1996

Safety and immunogenicity of investigational Shigella conjugate vaccines in Israeli volunteers.

The safety and immunogenicity of investigational conjugates, composed of the O-specific polysaccharides of Shigella sonnei and Shigella flexneri type 2a covalently bound to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), were evaluated in 192 Israeli soldiers. None had significant local reactions or fever. Fourteen days after injection, 90% of S. sonnei-rEPA recipients and 73 to 77% of S. flexneri-rEPA recipients had a fourfold or greater increase in serum immunoglobulin G (IgG) and IgA anti-lipopolysaccharide (anti-LPS) levels; at 2 years, these remained higher than at prevaccination (P < 0.01). There was a fourfold or greater increase in IgM anti-LPS in 20% of vaccinees at 2 weeks, but levels returned to prevaccination values at 6 to 12 months. IgG was the highest and most sustained class of LPS antibodies. Reinjection at day 42 did not boost antibody levels. Eighteen of 23 (78%) who received S. sonnei-rEPA and 13 of 19 (68%) who received S. flexneri-rEPA. had significant IgA-secreting cell responses. Significant IgG antibody-secreting cell responses were detected in 19 of 23 (83%) and 11 of 19 (58%) volunteers following vaccination with S. sonnei-rEPA and S. flexneri 2a-rEPA, respectively. On the basis of these data, further evaluation of the Shigella conjugates for protective efficacy in field trials in Israel was started.

Adolescent↗
PubMed1996

Synthesis, characterization, and immunological properties in mice of conjugates composed of detoxified lipopolysaccharide of Salmonella paratyphi A bound to tetanus toxoid with emphasis on the role of O acetyls.

Salmonella paratyphi A, the second most common cause of enteric fever in Southeast Asia, is a habitant of and a pathogen for humans only. Lipopolysaccharides (LPS) are both essential virulence factors and protective antigens for systemic infections caused by groups A, B, C, and D nontyphoidal salmonellae. The O-specific polysaccharide of S. paratyphi A is composed of a trisaccharide, -->2-alpha-D)-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Galp -(1-->, with a branch of D-paratose from the C-3 of alpha-D-mannose, and the C-3 of beta-L-rhamnose is partially O acetylated (C. G. Hellerqvist, B. Lindberg, K. Samuelsson, and A. A. Lindberg, Acta Chem. Scand. 25:955-961, 1971). On the basis of data from our investigational vaccines for enteric bacterial pathogens, including group B salmonellae (D. C. Watson, J. B. Robbins, and S. C. Szu, Infect. Immun. 60:4679-4686, 1992), conjugates composed of the detoxified LPS of S. paratyphi A bound to tetanus toxoid (TT) were prepared by several schemes. LPS was detoxified with acetic acid or with hydrazine; the latter removed O acetyls from the O-specific polysaccharide. The detoxified polysaccharides were activated with cyanogen bromide (CNBr) or with 1-cyano-4-dimethylaminopyridinium tetratfluoroborate (CDAP) and bound to TT with or without a spacer. Solutions of 2.5 microgram of saccharide, alone or as a conjugate, were injected subcutaneously into young mice, and LPS and TT antibodies were measured by enzyme-linked immunosorbent assaying. A conjugate synthesized with higher-molecular-weight O-SP elicited the highest anti-LPS levels. Only conjugates with O acetyls elicited serum immunoglobulin G anti-LPS with bactericidal activity. There were no statistically significant differences between LPS antibody levels elicited by conjugates synthesized with or without a spacer. The conjugate with O-specific polysaccharide activated by CDAP and bound to TT without a spacer elicited the highest level of TT antibodies. Clinical evaluation (if S. paratyphi A conjugates is planned.

Acetylation↗
PubMed1996

Safety and immunogenicity of Staphylococcus aureus type 5 capsular polysaccharide-Pseudomonas aeruginosa recombinant exoprotein A conjugate vaccine in patients on hemodialysis.

Seventeen volunteers with ESRD on hemodialysis, negative for infection with HIV or hepatitis B and C and not receiving immunosuppressive therapy, were injected two times 6 wk apart with 25 micrograms of Staphylococcus aureus Type 5 capsular polysaccharide-Pseudomonas aeruginosa exoprotein A (rEPA) conjugate. Controls were healthy adults, 18 to 44 yr old, injected previously with the same vaccine. None of the patients had fever or significant elevations in their SGOT or SGPT attributable to the vaccine. Two vaccinees had transient induration > 1 cm in diameter at the injection site. The preimmunization geometric mean (GM) Type 5 antibody levels of the ESRD patients and controls were similar. Type 5 antibody levels of the three major immunoglobulin (lg) classes rose at 2 and 6 wk after immunization (P < 0.001 for lgG, P < 0.005 for lgM, and P = 0.0001 for lgA). Reimmunization at 6 wk did not elicit a booster response. At 6 months, the GM lgG level of the patients was approximately 50% of that of the healthy volunteers and 14 of 17 had a more than fourfold higher antibody level than the preimmune value. The GM lgM level, in contrast, declined to the preimmunization value. Vaccine-induced Type 5 antibodies had opsonophagocytic activity. There was a slight increase of lgG antibodies to the heterologous S. aureus Type 8 polysaccharide (P < 0.01) that was sustained at 6 months. The S. aureus Type 5-rEPA vaccine is safe and immunogenic in ESRD patients, and evaluation of its effectiveness against S. aureus bacteremia in this at-risk group is planned.

ADP Ribose Transferases↗
PubMed1995

The solution structure of the human ETS1-DNA complex reveals a novel mode of binding and true side chain intercalation.

The solution structure of a 24.4 kDa specific complex of the DNA-binding domain (DBD) of the human ETS1 (hETS1) oncoprotein with a 17-mer DNA has been solved by NMR. The interaction of the hETS1 DBD with DNA reveals a surprising twist on the general features of helix-turn-helix (HTH)-DNA interactions. Major groove recognition involves the C-terminal two thirds of the HTH recognition helix, while minor groove recognition occurs via true intercalation of the side chain of Trp-28, which extends from the minor to the major groove. This results in a sharp kink of approximately 60 degrees and a widening of the minor groove over one-half turn of the DNA. The orientation of the HTH element of the hETS1 DBD with respect to the major groove is significantly rotated relative to other HTH proteins. These observations establish the ETS family of DNA-binding proteins as a distinct family of HTH proteins.

Amino Acid Sequence↗
PubMed1995

Identification of the peptide binding motif for HLA-B44, one of the most common HLA-B alleles in the Caucasian population.

Most peptides that bind to a particular MHC class I molecule share amino acid residues that are thought to physically "anchor" the peptide to polymorphic pockets within the class I binding site. Sequence analysis of endogenous peptides bound to HLA-B44 revealed two potential dominant anchor residues: Glu at P2 and Tyr, or occasionally Phe, at P9. In vitro assembly assays employing synthetic peptides and recombinant HLA-B44 produced by Escherichia coli revealed that an acidic amino acid at P2 was necessary for promoting stable peptide binding to HLA-B44. Surprisingly, although Tyr was almost exclusively found at P9 of the endogenous peptide sequences, a wide variety of amino acid residues such as Leu, Ala, Arg, Lys, His, and Phe could be tolerated at this position. Using this information, we identified antigenic peptides from the influenza virus components nonstructural protein 1 and nucleoprotein that are presented by HLA-B44 to antiinfluenza type A cytotoxic T lymphocytes. In addition, cytotoxic T lymphocytes induced by these antigenic peptides were shown to be capable of recognizing endogenously processed peptides from influenza-infected cells, indicating a potential use for these peptides in vaccine development. Finally, molecular models were created to investigate the possible ways in which the anchor residues might function to stabilize the binding of peptides to HLA-B44, and these models indicate that the acidic residue at P2 most likely interacts primarily with Lys 45 of the HLA-B44 heavy chain and makes additional contacts with Ser 67 and Tyr 9.

Alleles↗
PubMed1995

High-yield production of diphtheria toxin mutants by high-density culture of C7 (beta)tox+ strains grown in a non-deferrated medium.

A high-density growth approach was utilized to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheria: C7 (beta)(tox-201,tox-9) and C7 (beta)(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are CRM9 and CRM107; both of them carry the mutation in their binding site and, as a result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. Since iron inhibits diphtheria toxin production, the traditional approach has been to grow the bacteria in a very low iron concentration. The procedure described here involved the use of a modified, non-deferrated, growth medium that provided fast and high-density growth of the bacteria, and which, when associated with simultaneous depletion of glucose and iron, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15-20 g/l dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM products, which remained stable following microfiltration and ultrafiltration, could be easily purified using a two-step chromatography procedure.

Corynebacterium diphtheriae↗
PubMed1994

The HLA-B14 peptide binding site can accommodate peptides with different combinations of anchor residues.

Most peptides that bind to a particular major histocompatibility complex class I molecule share amino acid residues important for binding at one or two positions. Sequence analyses of peptides bound to HLA-B14 revealed at least four candidates for these so-called anchor residues: Arg at P2, Tyr at P3, Arg at P5, and Leu at P9. Combinations of any three of these amino acids sufficed for binding to HLA-B14 in vitro. Using this information, we identified an antigenic peptide critical for cytotoxic T lymphocyte recognition of virus-infected cells. Molecular models of HLA-B14 peptide complexes were constructed to investigate how the potential anchor residues might function. By using binding data to calculate the contribution to binding of each amino acid at anchor positions and predicting the stability of all possible nonapeptide complexes that could be formed from antigenic proteins, we estimate that three known antigenic nonapeptides are in the highest affinity cohort of peptides. Thus, even when multiple combinations of anchor residues contribute to binding, antigenic peptides are routinely identifiable.

Alanine↗
PubMed1994

The cooperative binding of chromosomal protein HMG-14 to nucleosome cores is reduced by single point mutations in the nucleosomal binding domain.

Mutants of human chromosomal protein HMG-14 were generated by site directed mutagenesis and used to study functional domains in this protein. A replacement of serine by cysteine at position 7 did not affect the binding of the protein to nucleosome cores. The sulfhydryl group in the nucleosome-bound protein is accessible to modifying agents suggesting that position 7 in the protein is not in close contact with either the DNA or the histones in the core particles. Under cooperative binding conditions, replacements of alanine by proline at position 21, or of lysine by cysteine at position 26, decreased the affinity of the protein for nucleosome cores 6.7- and 3-fold respectively. In contrast, the non-cooperative mode of binding was only minimally affected. A replacement of glutamic acid by glutamine at position 76 caused only minor changes in the binding of the protein to the cores. The results indicate that single point mutations, which change either the conformation or change in the nucleosomal binding domain of the protein, significantly reduce the ability of the HMG-14 protein to bind to nucleosome cores. We suggest that in chromatin the protein binds to nucleosomes in a cooperative manner and that upon binding to nucleosomes the protein acquires a distinct conformation.

Alanine↗