Mass spectrometry measurement of a therapeutic peptide for use in multiple sclerosis.

Multiple sclerosis is an autoimmune disease of the central nervous system believed to be mediated by pathogenic T lymphocytes. We have developed a next-generation therapy in which cells secrete specific therapeutic molecules to silence these aberrant T cells. We have shown that fibroblasts, transduced to secrete a myelin basic protein-derived peptide, abrogate disease in the murine experimental autoimmune encephalomyelitis model of multiple sclerosis, which we hypothesized using a low-zone tolerance mechanism. To determine the efficacy (or not) of this therapy in humans, we must ensure that patients receive comparable doses of therapeutic peptide. To this end, we have used liquid chromatography coupled to tandem mass spectrometry to detect a tryptic peptide, derived from the secreted therapeutic product, at nanomolar concentrations. Success depended on growing the transduced fibroblasts in defined PC-1 medium in the presence of a cocktail of protease inhibitors.

Cell-based gene therapy experiments in murine experimental autoimmune encephalomyelitis.

With the ultimate goal of developing a novel treatment for multiple sclerosis (MS), we have developed a cell-based gene therapy protocol for the treatment of murine experimental autoimmune encephalomyelitis (EAE), a powerful animal model for MS. We have determined that transduced fibroblasts secreting encephalogenic epitopes, when injected into mice with EAE, cause a striking abrogation of disease. Both myelin basic protein (MBP) and proteolipid protein mini-gene constructs expressed in syngeneic fibroblast cells were capable of ameliorating ongoing EAE induced by MBP protein. These experiments are crucial since they suggest that not all encephalogenic epitopes need be secreted for the control of disease. We also demonstrate the success of this protocol when transduced syngeneic, and most importantly, allogeneic cells are sequestered within an implantable chamber. Furthermore, we find that through modifying antigen expression by changing the signal sequence of the mini-gene construct, we were able to significantly reduce the dose of cells required for treatment. These improvements to the mini-gene delivery system are critical for the eventual translation of our protocol to the clinic.

H-2Ld-alloreactive T cell hybridomas utilize diverse V alpha and V beta T cell receptor chains.

We have sequenced the TCRs from Ld-specific alloreactive T cell hybridomas, whose reactivities we have found to be quite representative of those of a primary dm2 anti-BALB/cJ mixed lymphocyte reaction. We find V beta 6, V beta 7, V beta 8 and V beta 10 gene segments. V alpha usage is diverse, although closely related to that from peptide-specific Ld-restricted CTLs. V alpha-V beta selection provides evidence of preferential pairing. Amino acid frequency analysis shows that the alpha CDR2 region is rich in charged amino acids, in contrast to the beta CDR2 region. Our data suggests the beta chain may be more immunoglobulin-like than the alpha chain, and that charge complementarity may be important in TCR-MHC interactions. We do not consider our results to be contradictory to those previously reported but rather they may represent an early, more diverse response.

Cofilin is an essential component of the yeast cortical cytoskeleton.

We have biochemically identified the Saccharomyces cerevisiae homologue of the mammalian actin binding protein cofilin. Cofilin and related proteins isolated from diverse organisms are low molecular weight proteins (15-20 kD) that possess several activities in vitro. All bind to monomeric actin and sever filaments, and some can stably associate with filaments. In this study, we demonstrate using viscosity, sedimentation, and actin assembly rate assays that yeast cofilin (16 kD) possesses all of these properties. Cloning and sequencing of the S. cerevisiae cofilin gene (COF1) revealed that yeast cofilin is 41% identical in amino acid sequence to mammalian cofilin and, surprisingly, has homology to a protein outside the family of cofilin-like proteins. The NH2-terminal 16kD of Abp1p, a 65-kD yeast protein identified by its ability to bind to actin filaments, is 23% identical to yeast cofilin. Immunofluorescence experiments showed that, like Abp1p, cofilin is associated with the membrane actin cytoskeleton. A complete disruption of the COF1 gene was created in diploid cells. Sporulation and tetrad analysis revealed that yeast cofilin has an essential function in vivo. Although Abp1p shares sequence similarity with cofilin and has the same distribution as cofilin in the cell, multiple copies of the ABP1 gene cannot compensate for the loss of cofilin. Thus, cofilin and Abp1p are structurally related but functionally distinct components of the yeast membrane cytoskeleton.

Yeast proteins associated with microtubules in vitro and in vivo.

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

A small basic ribosomal protein from the extreme thermophilic archaebacterium Sulfolobus solfataricus that has no equivalent in Escherichia coli.

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene has been confirmed by partial amino acid sequencing. The protein shows no sequence similarity to any of the ribosomal proteins from eubacteria (Escherichia coli) or to those that have been reported from eukaryotes.

A small basic ribosomal protein in Sulfolobus solfataricus equivalent to L46 in yeast: structure of the protein and its gene.

The structure of the gene for a small, very basic ribosomal protein in Sulfolobus solfataricus has been determined and the structure of the protein coded by this gene (L46e) has been confirmed by partial amino acid sequencing. The protein shows substantial sequence homology to the eukaryotic ribosomal proteins L39 in rat and L46 in yeast. There is no sequence homology to any of the eubacterial ribosomal proteins suggesting that this protein is absent in the eubacterial ribosome.

The primary structure of the ribosomal A-protein (L12) from the halophilic eubacterium Haloanaerobium praevalens.

The ribosomal A-protein, equivalent to the ribosomal protein L12 from Escherichia coli, has been sequenced from the anaerobic halophilic eubacterium Haloanaerobium praevalens (DSM 2228). The protein contains 122 amino acids, has a composition of Asp6, Asn2, Thr2, Ser6, Glu22, Pro2, Gly13, Ala19, Val12, Met4, Ile5, Leu11, Phe3, Lys14, Arg1 and has a molecular weight of 12,691. The hydrophilicity profile was determined for this protein. A phylogenetic or cluster tree was calculated from computer analysis of the sequence data on eubacterial ribosomal A-proteins. H. praevalens clusters with a group that includes Bacillus subtilis, Micrococcus lysodeikticus, Bacillus stearothermophilus and Clostridium pasteurianum.

The complete amino acid sequence of the ribosomal 'A' protein (L12) from Bacillus stearothermophilus.

The complete amino acid sequence of the ribosomal 'A' protein (Bst L12) has been determined from Bacillus stearothermophilus. The protein contains 122 amino acids and has a composition of Asp4, Ans3, Thr6, Glu20, Gln2, Pro3, Gly9, Ala23, Val13, Met2, Ile11, Leu8, Phe2, Lys15, Arg1 and a molecular mass of 12737 Da.