Link between Organ-specific Antigen Processing by 20S Proteasomes and CD8+ T Cell–mediated Autoimmunity

Adoptive transfer of cross-reactive HSP60-specific CD8+ T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8+ T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of α and β chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8+ T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses

Gas Phase Thermal Denaturation of an Oligonucleotide Duplex and Its Complexes with Minor Groove Binders

Electrospray ionization with in-source collisionally induced dissociation has been used to probe the gas phase stability of an oligonucleotide duplex and its complexes with some minor groove binding drugs. On the basis of the arguments developed in detail by Drahos et al. (J. Mass Spectrom. 1999; 34:1373), this type of experiment can also be described as 'thermal denaturation in the gas phase'. We found that the gas phase denaturation curves were very similar to the solution phase denaturation curves determined by the traditional UV spectrophotometric method and, by analogy with the melting temperature T(m) which characterizes the stability in solution, we define a melting voltage V(m) to characterize the stability in the gas phase. A comparison of the T(m) and V(m) relative values suggests that the structure of the complexes is conserved during the electrospray process which transfers the ions from the solution to the gas phase

Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function

Tough gel adhesive is an effective method for meniscal repair in a bovine cadaveric study

Abstract Purpose To test tough gel adhesives to repair meniscus tears under relevant loading conditions and determine if they have adequate biomechanical properties to repair meniscus tears in a bovine cadaveric study. Methods Cyclic compression tests on 24 dissected bovine knees were performed. The tough gel adhesive was used either as an adhesive patch or as a coating bonded onto commercially available surgical sutures. Forty‐eight menisci were tested in this study; 24 complete radial tears and 24 bucket‐handle tears. After preconditioning, the specimens underwent 100 cycles of compression, (800 N/0.5 Hz) on an Instron© machine and the size of the gaps measured. One third of the menisci were repaired with pristine sutures, one third with adhesive patches, and one third with sutures coated in adhesive gel. The size of the gaps was compared after 100 and 500 cycles of compression. Results The mean gap measured at the tear site without treatment was 6.46 mm (± 1.41 mm) for radial tears and 1.92 mm (± 0.65 mm) for bucket‐handle tears. After treatment and 500 cycles of compression, the mean gap was 1.63 mm (± 1.41 mm) for pristine sutures, 1.50 mm (± 1.16 mm) for adhesive sutures and 2.06 mm (± 1.53 mm) for adhesive gel patches. There was no significant difference between treatments regardless of the type of tear. Also, the gaps for radial tears increased significantly with the number of compression cycles applied (p > 0.001). Conclusion From a biomechanical standpoint, the tough adhesive gel patch is as effective as suturing. In addition, it would allow the repair of non‐suturable tears and thus broaden the indications for meniscus repair. Level of evidence Controlled laboratory study

Detection and identification of tumorassociated protein variants in human hepatocellular carcinomas

The proteomic approach is a valuable tool to detect and identify proteins that are associated with cancer. In previous investigations on experimentally induced rat hepatomas, we detected aldose reductase-like protein (ARLP) as a highly significant marker protein. Our present study was intended to look for the presence of similar tumor-associated marker proteins on human hepatocellular carcinomas (HCC). We found several novel tumorassociated protein variants that represent members of the aldo-keto reductase (AKR) superfamily. Human aldose reductase-like protein-1 (hARLP-1) was the most prominent tumorassociated AKR member detected in HCC by 2-dimensional electrophoresis (2-DE) and identified by mass spectrometric fingerprinting. The enzyme was found in 4 distinct forms (hARLP-1, 36/7.4 (kd/pI); hARLP-2, 36/7.2; hARLP-3, 36/6.4; and hARLP-4, 33/7.35). In addition, a human aldose reductase-like protein (hARLP-5, 36/7.6) was identified that differed from hARLP-1 by 1 amino acid (D313N), indicating 2 allelic forms of the human aldose reductase-like gene. A novel antibody directed against common parts of the hARLPs revealed hARLP reactivity in human HCC by immunohistochemistry. Furthermore, aldose reductase (AR) was identified and characterized as a tumor-associated variant. In conclusion