Conjugation of synthetic peptides to proteins: quantitation from S-carboxymethylcysteine released upon acid hydrolysis

A method described here for conjugating synthetic peptides to carrier proteins provides a convenient method for determining peptide-to-carrier protein ratios. N-Bromoacetyl-containing peptides are reacted in situ with carrier proteins in which the disulfide bonds were reduced with tri-n-butylphosphine. At pH 7-8 and ambient temperature, the newly formed sulfhydryl groups of the carrier protein react exclusively with the bromoacetyl mokiety of the peptide to form conjugates having stable thio ether linkages. Acid hydrolyses of these conjugates release S-carboxymethylcysteine in amounts proportional to the amounts of peptides conjugated and thus allow determination of peptide-to-protein ratios.     

Isolation and characterization of monkey interphotoreceptor retinoid-binding protein, a unique extracellular matrix component of the retina

The interphotoreceptor retinoid-binding protein (IRBP) has been isolated from monkey interphotoreceptor matrix (IPM). Following gentle washing of the IPM from the retinal surface, the protein was purified to homogeneity by concanavalin A-Sepharose affinity chromatography, ion-exchange high-performance liquid chromatography (HPLC), and size-exclusion HPLC. Bovine IRBP was purified similarly and compared with the monkey protein. Sedimentation equilibrium analysis yielded a molecular weight of 106 000 +/- 2900 for the native monkey protein. Sedimentation velocity analysis gave a sedimentation coefficient of 5.4 +/- 0.3 S and a frictional ratio of 1.59, indicating an asymmetrical molecular shape. IRBP contains neutral sugar, including fucose, and sialic acid; the glycoprotein nature of the proteins probably accounts for the microheterogeneity observed in the electrofocusing pattern of both bovine and monkey IRBP. Both IRBPs have isoelectric points between 6.0 and 7.0. The fluorescence emission lambda max of the bound ligand was 470 nm with excitation at 340 nm, while the excitation lambda max was 333 nm with emission at 470 nm, for monkey IRBP incubated with exogenous all-trans-retinol. The amino acid compositions of the monkey and bovine proteins are similar; nonpolar amino acids account for over 50% of the residues, which may explain the apparent hydrophobic nature of the isolated proteins. The amino-terminal analyses indicated considerable homology between the monkey and bovine IRBPs in this region and verified the purity of the isolated proteins. IRBP thus appears to be a unique, conserved glycoprotein of the retinal extracellular matrix that could serve as a retinoid-transport vehicle.     

Restricted heterogeneity of antibody to gp120 and p24 in AIDS

Neurologic complications and cerebrospinal fluid (CSF) abnormalities are common in AIDS. We found that a substantial number of AIDS patients with neurologic involvement had oligoclonal IgG bands in CSF and sera by IEF. Using an IEF-Ag overlay technique, anti-gp120 antibody activity was demonstrated more frequently than anti-p24 antibody activity. These antibody activities exhibited restricted heterogeneity of their IEF pattern; this restriction may contribute to the relatively low titers of neutralizing antibody found in AIDS sera. None of the CSF and serum oligoclonal bands showed anti-HIV antibody activity, suggesting that they are directed against opportunistic agents or result from immunodysregulation.     

Location of the CD8 T Cell Epitope within the Antigenic Precursor Determines Immunogenicity and Protection against the Toxoplasma gondii Parasite

CD8 T cells protect the host from disease caused by intracellular pathogens, such as the Toxoplasma gondii (T. gondii) protozoan parasite. Despite the complexity of the T. gondii proteome, CD8 T cell responses are restricted to only a small number of peptide epitopes derived from a limited set of antigenic precursors. This phenomenon is known as immunodominance and is key to effective vaccine design. However, the mechanisms that determine the immunogenicity and immunodominance hierarchy of parasite antigens are not well understood.Here, using genetically modified parasites, we show that parasite burden is controlled by the immunodominant GRA6-specific CD8 T cell response but not by responses to the subdominant GRA4- and ROP7-derived epitopes. Remarkably, optimal processing and immunodominance were determined by the location of the peptide epitope at the C-terminus of the GRA6 antigenic precursor. In contrast, immunodominance could not be explained by the peptide affinity for the MHC I molecule or the frequency of T cell precursors in the naive animals. Our results reveal the molecular requirements for optimal presentation of an intracellular parasite antigen and for eliciting protective CD8 T cells.     

Frequent Seizures Are Associated with a Network of Gray Matter Atrophy in Temporal Lobe Epilepsy with or without Hippocampal Sclerosis

Objective

Patients with temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) have diffuse subtle gray matter (GM) atrophy detectable by MRI quantification analyses. However, it is not clear whether the etiology and seizure frequency are associated with this atrophy. We aimed to evaluate the occurrence of GM atrophy and the influence of seizure frequency in patients with TLE and either normal MRI (TLE-NL) or MRI signs of HS (TLE-HS).

Methods

We evaluated a group of 172 consecutive patients with unilateral TLE-HS or TLE-NL as defined by hippocampal volumetry and signal quantification (122 TLE-HS and 50 TLE-NL) plus a group of 82 healthy individuals. Voxel-based morphometry was performed with VBM8/SPM8 in 3T MRIs. Patients with up to three complex partial seizures and no generalized tonic-clonic seizures in the previous year were considered to have infrequent seizures. Those who did not fulfill these criteria were considered to have frequent seizures.

Results

Patients with TLE-HS had more pronounced GM atrophy, including the ipsilateral mesial temporal structures, temporal lobe, bilateral thalami and pre/post-central gyri. Patients with TLE-NL had more subtle GM atrophy, including the ipsilateral orbitofrontal cortex, bilateral thalami and pre/post-central gyri. Both TLE-HS and TLE-NL showed increased GM volume in the contralateral pons. TLE-HS patients with frequent seizures had more pronounced GM atrophy in extra-temporal regions than TLE-HS with infrequent seizures. Patients with TLE-NL and infrequent seizures had no detectable GM atrophy. In both TLE-HS and TLE-NL, the duration of epilepsy correlated with GM atrophy in extra-hippocampal regions.

Conclusion

Although a diffuse network GM atrophy occurs in both TLE-HS and TLE-NL, this is strikingly more evident in TLE-HS and in patients with frequent seizures. These findings suggest that neocortical atrophy in TLE is related to the ongoing seizures and epilepsy duration, while thalamic atrophy is more probably related to the original epileptogenic process.     

The Role of Half-Transporters in Multidrug Resistance

ATP-binding cassette proteins comprise a superfamily of transporter proteins, a subset of which have been implicated in multidrug resistance. Although P-glycoprotein was described over 15 years ago, the recent expansion in the number of transporters identified has prompted renewed interest in the role of drug transporters in clinical drug resistance. These newly identified transporters include additional members of the MRP family, ABC2, and a new half-transporter, MXR/BCRP/ABCP1. This half-transporter confers high levels of resistance to mitoxantrone, anthracyclines, and the camptothecins SN-38 and topotecan. At 72 kDa, MXR localizes to the plasma membrane in cells which highly overexpress the protein either through gene amplification or though gene rearrangement. Future studies will be aimed at identifying an inhibitor, and attempting to translate recognition of this new transporter into a target for anticancer treatment.     

Variation in Resistance to High Hydrostatic Pressure and rpoS Heterogeneity in Natural Isolates of Escherichia coli O157:H7

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.     

Selective and facile cyclization of N-chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems.

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72, 857-867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N-chloroacetylated, C-cysteine amide peptides from the C4 domain of HIV-1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed-phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS-PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.