The model B6(dom1) minor histocompatibility antigen is encoded by a mouse homolog of the yeast STT3 gene

The B6(dom1) minor histocompatibility antigen (MiHA) is a model antigen, since it is both the epitome of an immunodominant epitope and an ideal target for adoptive cancer immunotherapy. Based on DNA sequencing and MS/MS analyses, we report that B6(dom1) corresponds to amino acids 770-778 (KAPDNRETL) of a protein we propose to call SIMP (source of immunodominant MHC-associated peptides) that is encoded by a mouse homolog of the yeast STT3gene. STT3, a member of the oligosaccharyltransferase complex, is essential for cell proliferation. Phenotypic and genotypic analyses among eight strains of mice revealed a precise correlation between susceptibility or resistance to B6(dom1)-specific cytotoxic T lymphocytes (CTLs) and the presence of a Glu vs Asp amino acid at position 776 of the SIMP protein, respectively. Strikingly, while the difference in the amino acid sequence 770-778 encoded by the two SIMP alleles represents a very conservative substitution, these allelic peptides were not crossreactive at the CTL level, and both peptides were immunodominant when presented to mice homozygous for the opposite allele. In addition, we have cloned a human ortholog of SIMP whose predicted protein shares 97% amino acid identity with mouse SIMP. These results strengthen the concept that MHC class-I-associated MiHAs originate as a consequence of rare polymorphisms among highly conserved genes. Furthermore, the notion that a peptide differing from a self analog by a single methylene group can be immunodominant has implications regarding our understanding of the mechanisms of immunodominance.     

HPLC-MS/MS analysis of the products generated from all-trans-retinoic acid using recombinant human CYP26A

Two mammalian hCYP26A expression systems have been used to analyze the metabolic products of CYP26A. Through the use of extensive HPLC, UV spectroscopy, and liquid chromatography/tandem mass spectrometry (LC-MS/MS) methodology, we have conclusively demonstrated that the complex mixture of products comprises 4-OH-all-trans-retinoic acid, 4-oxo-all-trans-retinoic acid, and 18-OH-all-trans-retinoic acid, and more polar products, partially identified as dihydroxy and mono-oxo, mono-hydroxy derivatives. These more polar products are presumed to result from multiple hydroxylations on the beta-ionone ring. The inter-relationship of initial and polar metabolites was inferred from both gene-dose and time-course experiments. Both initial and secondary metabolic steps after 4-oxo-all-trans-retinoic acid are ketoconazole-sensitive, suggesting that steps in the production of water-soluble metabolites are cytochrome P450-dependent.     

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug,isoniazid

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.     

A novel two-step mechanism for removal of a mitochondrial signal sequence involves the mAAA complex and the putative rhomboid protease Pcp1

The yeast protein cytochrome c peroxidase (Ccp1) is nuclearly encoded and imported into the mitochondrial intermembrane space, where it is involved in degradation of reactive oxygen species. It is known, that Ccp1 is synthesised as a precursor with a N-terminal pre-sequence, that is proteolytically removed during transport of the protein. Here we present evidence for a new processing pathway, involving novel signal peptidase activities. The mAAA protease subunits Yta10 (Afg3) and Yta12 (Rca1) were identified both to be essential for the first processing step. In addition, the Pcp1 (Ygr101w) gene product was found to be required for the second processing step, yielding the mature Ccp1 protein. The newly identified Pcp1 protein belongs to the rhomboid-GlpG superfamily of putative intramembrane peptidases. Inactivation of the protease motifs in mAAA and Pcp1 blocks the respective steps of proteolysis. A model of coupled Ccp1 transport and N-terminal processing by the mAAA complex and Pcp1 is discussed. Similar processing mechanisms may exist, because the mAAA subunits and the newly identified Pcp1 protein belong to ubiquitous protein families.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

Detection of the embryotoxic potential of cyclophosphamide by using a combined system of metabolic competent cells and embryonic stem cells

In order to develop a method for detecting metabolism-mediated embryotoxicity, differentiating embryonic stem (ES) cells were exposed to the well-known proteratogen, cyclophosphamide (CPA). CPA was tested in a scientifically validated embryonic stem-cell test (EST), and in the newly developed reporter-gene assay for developmental cardiotoxicity. Both assays gave false-negative results. Because no metabolic competence (cytochrome P450 [CYP] activity) was found in the ES cells under the selected culture conditions, a simple biotransformation system was combined with the reporter-gene assay. As the metabolic pathway of CPA is well characterised, the genetically engineered mammalian cell line V79, transfected with CYP2B1 cDNA, was selected as a biotransformation system. CYP2B1 is responsible for transforming CPA into teratogenically active metabolites. The supernatants of genetically engineered V79 cells were analysed in the reporter-gene assay for developmental cardiotoxicity. In preliminary experiments, the combined system was able to detect the embryotoxic potential of the proteratogen, CPA.     

Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones

Erythropoietin (EPO) is normally present in urine at a low concentration (about 1IU/L, i.e., about 10ng/L) for a total protein concentration of at least 50mg/L. A method to study the isoelectric profile of this hormone from 20-ml urine aliquots without previous purification was developed. This method involves isoelectric focusing of the retentate from ultrafiltered urine. Both the ultrafiltration and the isoelectric focusing required precautionary measures to prevent EPO degradation by the proteases that are present in urine. Because classical immunoblotting gave rise to an unspecific detection of various urinary proteins in the focused retentate, it was essential to use the "double-blotting" process developed to solve this problem. Sufficient sensitivity was achieved using amplified chemiluminiscent detection after the blotting membrane was treated with dithiotreitol. The patterns that were revealed from various urinary samples proved to be highly heterogeneous as they were composed of more than 10 isoforms in a pI range of 3.7-4.7. Clear transformation of the patterns was observed in the case of treatment by the recombinant hormone, suggesting that this method can be regarded an efficient tool for indicating recombinant EPO misuse in sports. It may also open new investigations in the field of physiologic or pathologic exploration.     

Novel locus required for expression of high-level macrolide-lincosamide-streptogramin B resistance in Staphylococcus aureus

The yycF1(Ts) mutation in Staphylococcus aureus conferred hypersensitivity to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics on strains either containing or lacking ermB. The overexpression of the S. aureus Ssa protein restored the yycF1 mutant to wild-type levels of susceptibility. Inactivation of ssa in an unmutagenized strain dramatically reduced ermB-based resistance. Conditional loss of function or expression of ssa in the yycF1 mutant is proposed to result in the observed hypersensitivity to MLS(B) antibiotics.     

Inhibition of antigen-specific T cell proliferation and cytokine production by protein kinase A type I

cAMP inhibits biochemical events leading to T cell activation by triggering of an inhibitory protein kinase A (PKA)-C-terminal Src kinase pathway assembled in lipid rafts. In this study, we demonstrate that activation of PKA type I by Sp-8-bromo-cAMPS (a cAMP agonist) has profound inhibitory effects on Ag-specific immune responses in peripheral effector T cells. Activation of PKA type I inhibits both cytokine production and proliferative responses in both CD4(+) and CD8(+) T cells in a concentration-dependent manner. The observed effects of cAMP appeared to occur endogenously in T cells and were not dependent on APC. The inhibition of responses was not due to apoptosis of specific T cells and was reversible by a PKA type I-selective cAMP antagonist. This supports the notion of PKA type I as a key enzyme in the negative regulation of immune responses and a potential target for inhibiting autoreactive T cells.