Identification of a mutated receptor-like protein tyrosine phosphatase kappa as a novel,class II HLA-restricted melanoma antigen

Recent studies increasingly point to a pivotal role of CD4(+) T cells in human anti-tumor immune response. Here we show that lymphocytes purified from a tumor-infiltrated lymph node of a melanoma patient that had remained disease free for 10 years after surgical resection of a lymph node metastasis comprised oligoclonal class II HLA-restricted CD4(+) T cells recognizing the autologous tumor cells in vitro. In fact, the CD4(+) T cell clones isolated from these lymphocytes displayed a tumor-specific, cytotoxic activity in addition to a Th1-like cytokine profile. By a genetic approach, a peptide derived from a mutated receptor-like protein tyrosine phosphatase kappa was identified as a novel HLA-DR10-restricted epitope for all the melanoma-specific CD4(+) T cell clones. The immunogenic peptide was shown to contain the mutated residue that was crucial for T cell recognition and activation. Moreover, a systemic immunity against the mutated peptide was detectable in the patient's peripheral blood T lymphocytes obtained during the disease-free period of follow-up. These findings further support the relevance of CD4(+) T cells directed against mutated epitopes in tumor immunity and provide the rationale for a possible usage of mutated, tumor-specific Ags for immunotherapy of human cancer.     

The Duplex-Harpin Conformational Transition of d(CGCGCGATCGCGCG) and d(CGCGCGTACGCGCG): A Thermodynamic and Kinetic Study

Abstract

We have studied the duplex-hairpin conformational transition in two perfectly palindromic sequences, d(CGCGCGATCGCGCG)(I) and d(CGCGCGTACGCGCG)(II), by means of UV-melting, electrophoretic and T-jump experiments. Both tetradecamers exhibit biphasic thermal profiles. The lower temperature transition is concentration dependent whereas the higher temperature transition is not. The former transition has been characterized by gel electrophoresis and shows two distinct bands, whose intensity depends on temperature. This behavior is due to the occurrence of a slow premelting interconversion between the duplex and hairpin forms in both tetradecamers. The kinetics of hairpin formation from the duplex is studied by T-jump experiments. Relaxation spectra are well reproduced by a single relaxation time with rate constants characterized by a high temperature coefficient. In 10 mM NaCl, the duplex-hairpin conversion of I is characterized by an apparent activation energy of 96 ± 6 kcal/mol, a value rather close to the expected denaturation enthalpy. In 1 mM NaCl a value slightly lower has been obtained. The rate of duplex-hairpin interconversion has been found to decrease as the salt concentration is raised. These data suggest that the transformation from the duplex to the hairpin form should imply a transition state with a simultaneous breaking of most base pairs, if not total strand separation.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.     

The transcriptional landscape of the deep-sea bacterium Photobacterium profundum in both a toxR mutant and its parental strain

Background

Mutans streptococci are a group of gram-positive bacteria including the primary cariogenic dental pathogen Streptococcus mutans and closely related species. Two component systems (TCSs) composed of a signal sensing histidine kinase (HK) and a response regulator (RR) play key roles in pathogenicity, but have not been comparatively studied for these oral bacterial pathogens.

Results

HKs and RRs of 8 newly sequenced mutans streptococci strains, including S. sobrinus DSM20742, S. ratti DSM20564 and six S. mutans strains, were identified and compared to the TCSs of S. mutans UA159 and NN2025, two previously genome sequenced S. mutans strains. Ortholog analysis revealed 18 TCS clusters (HK-RR pairs), 2 orphan HKs and 2 orphan RRs, of which 8 TCS clusters were common to all 10 strains, 6 were absent in one or more strains, and the other 4 were exclusive to individual strains. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. While TCS complements were comparable within the six S. mutans strains, S. sobrinus DSM20742 lacked TCSs possibly involved in acid tolerance and fructan catabolism, and S. ratti DSM20564 possessed 3 unique TCSs but lacked the quorum-sensing related TCS (ComDE). Selected computational predictions were verified by PCR experiments.

Conclusions

Differences in the TCS repertoires of mutans streptococci strains, especially those of S. sobrinus and S. ratti in comparison to S. mutans, imply differences in their response mechanisms for survival in the dynamic oral environment. This genomic level study of TCSs should help in understanding the pathogenicity of these mutans streptococci strains.     

Heterochromatin,HP1 and methylation at lysine 9 of histone H3 in animals

We show that methylated lysine 9 of histone H3 (Me9H3) is a marker of heterochromatin in divergent animal species. It localises to both constitutive and facultative heterochromatin and replicates late in S-phase of the cell cycle. Significantly, Me9H3 is enriched in the inactive mammalian X chromosome (Xi) in female cells, as well as in the XY body during meiosis in the male, and forms a G-band pattern along the arms of the autosomes. Me9H3 is a constituent of imprinted chromosomes that are repressed. The paternal and maternal pronuclei in one-cell mouse embryos show a striking non-equivalence in Me9H3: the paternal pronucleus contains no immunocytologically detectable Me9H3. The levels of Me9H3 on the parental chromosomes only become equivalent after the two-cell stage. Finally, we provide evidence that Me9H3 is neither necessary nor sufficient for localisation of heterochromatin protein 1 (HP1) to chromosomal DNA.     

Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore

In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96 microM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.     

Improved Planning Abilities in Binge Eating

Objective

The role of planning in binge eating episodes is unknown. We investigated the characteristics of planning associated with food cues in binging patients. We studied planning based on backward reasoning, reasoning that determines a sequence of actions back to front from the final outcome.

Method

A cross-sectional study was conducted with 20 healthy participants, 20 bulimia nervosa (BN), 22 restrictive (ANR) and 23 binging anorexia nervosa (ANB), without any concomitant impulsive disorder. In neutral/relaxing, binge food and stressful conditions, backward reasoning was assessed with the Race game, promotion of delayed large rewards with an intertemporal discounting task, attention with the Simon task, and repeating a dominant behavior with the Go/No-go task.

Results

BN and to a lower extent ANB patients succeeded more at the Race game in food than in neutral condition. This difference discriminated binging from non-binging participants. Backward reasoning in the food condition was associated with lower approach behavior toward food in BN patients, and higher food avoidance in ANB patients. Enhanced backward reasoning in the food condition related to preferences for delayed large rewards in BN patients. In BN and ANB patients the enhanced success rate at the Race game in the food condition was associated with higher attention paid to binge food.

Conclusion

These findings introduce a novel process underlying binges: planning based on backward reasoning is associated with binges. It likely aims to reduce craving for binge foods and extend binge refractory period in BN patients, and avoid binging in ANB patients. Shifts between these goals might explain shifts between eating disorder subtypes.     

Autophosphorylation at the regulatory β subunit reflects the supramolecular organization of protein kinase CK2

Among the features of protein kinase CK2, autophosphorylation at its β-subunit(s) upon incubation with ATP/Mg⁺⁺ was early detected as a rapid and stoichiometric event occurring through an intramolecular mechanism as judged from kinetic analyses. The autophosphorylation site was mapped to Ser2 and, to a lesser extent, Ser3 both fulfilling the CK2 consensus sequence (MSSSEEV). The crystal structure of the heterotetrameric holoenzyme, however, is not compatible with an intramolecular autophosphorylation of the N-terminal stretch of either of the two β subunits. Here we show that efficient “intramolecular” autophosphorylation of the β subunit is crucially dependent on the formation of oligomers composed by several holoenzyme heterotetrameric protomers. Increasing ionic strength of the incubation medium promoting dissociation of the supramolecular oligomers abrogates β subunit autophosphorylation, although CK2 catalytic activity, as judged from the phosphorylation of exogenous substrates, is still quite evident. These findings, in conjunction with graphic modelization, support the view that CK2 autophosphorylation at its β subunits takes place through an “intraoligomeric” mechanism where the β subunits of a protomer are phosphorylated by the catalytic subunits of another adjacent protomer. It appears therefore that in vivo β autophosphorylation is symptomatic of supramolecular CK2 oligomers.     

LIMPIC: a computational method for the separation of protein MALDI-TOF-MS signals from noise

Background  

Mass spectrometry protein profiling is a promising tool for biomarker discovery in clinical proteomics. However, the development of a reliable approach for the separation of protein signals from noise is required. In this paper, LIMPIC, a computational method for the detection of protein peaks from linear-mode MALDI-TOF data is proposed. LIMPIC is based on novel techniques for background noise reduction and baseline removal. Peak detection is performed considering the presence of a non-homogeneous noise level in the mass spectrum. A comparison of the peaks collected from multiple spectra is used to classify them on the basis of a detection rate parameter, and hence to separate the protein signals from other disturbances.