Expansion of the antigenic repertoire of a single T cell receptor upon T cell activation.

Activated T cells and their naive precursors display different functional avidities for peptide/MHC, but are thought to have identical antigenic repertoires. We show that, following activation with a cognate mimotope (NRP), diabetogenic CD8(+) T cells expressing a single TCR (8.3) respond vigorously to numerous peptide analogs of NRP that were unable to elicit any responses from naive 8.3-CD8(+) T cells, even at high concentrations. The NRP-reactive, in vivo activated CD8(+) cells arising in pancreatic islets of nonobese diabetic mice are similarly promiscuous for peptide/MHC, and paradoxically this promiscuity expands as the aviditiy of the T cell population for NRP/MHC increases with age. Thus, activation and avidity maturation of T lymphocyte populations can lead to dramatic expansions in the range of peptides that elicit functional T cell responses.     

The Ig light chain restricted B6.kappa(-)lambda(SEG) mouse strain suggests that the IGL locus genomic organization is subject to constant evolution

In laboratory mice, the different Ig lambda light chain subtypes (lambda 1, lambda 2, lambda x and lambda 3) are expressed on 60, 16, 16 and 8%, respectively, of the lambda-positive peripheral B cells. Eighteen years ago, our laboratory characterized a lambda 1(-) wild mouse strain: SPE ( Mus spretus). In this report, we describe the characterization of another wild-derived Mus spretus inbred strain, SEG, that presents the same characteristic, namely the absence of lambda 1 expression. An almost congenic strain, B6.lambda(SEG), was detected in a series of recombinant congenic strains carrying 2% of SEG/Pas genome in a C57BL/6J background. This B6.lambda(SEG) strain was crossed to Igh (a) C kappa (-) mice in order to derive two different additional congenic strains: B6.kappa(-)lambda(SEG) Igh (a) and B6.kappa(-)lambda(SEG) Igh (b). In this paper, we characterize the genomic organization and the expression of the SEG IGL locus. Altogether, our data show that the SEG IGL locus is constituted by a single functional IGLJ2SEG-IGLC2SEG, two pseudo IGLJ4SEG1/2-IGLC4SEG1/2 gene clusters and two V gene segments: IGLV2SEG and IGLVXSEG. In particular, we show the absence of IGLV1 and IGLVSD26 gene segments. IGLVSD26 was reported to be present in some Mus m. musculus mice and absent in BALB/c. Here, we confirm its presence not only in other Mus m. musculus mice but also in Mus spretus mice. Consequently, we propose that IGLVSD26-related gene segments define a new family that we name V lambda 4. The study of the organization of different IGL loci, in addition to the V lambda 4(+) reported here, could elucidate questions concerning the evolution of the lambda locus.     

Involvement of polyamines in root development at low temperature in the subantarctic cruciferous species Pringlea antiscorbutica

Polyamine involvement in root development at low temperature was studied in seedlings of Pringlea antiscorbutica R. Br. This unique endemic cruciferous species from the subantarctic zone is subjected to strong environmental constraints and shows high polyamine contents. In the present study, free polyamine levels were modified by inhibitors of polyamine biosynthesis (D-arginine, difluoromethylornithine, cyclohexylammonium, and methylglyoxal-bis-guanylhydrazone) and variations of the endogenous pools were compared to changes in root growth. The arginine decarboxylase pathway, rather than that of ornithine decarboxylase, seemed to play a major role in polyamine synthesis in Pringlea antiscorbutica seedlings. Root, but not shoot, phenotypes were greatly affected by these treatments, which modified polyamine endogenous levels according to their expected effects. A positive correlation was found between agmatine level and growth rate of the primary root. Spermidine and spermine contents also showed positive correlations with primary root growth whereas the putrescine level showed neutral or negative effects on this trait. Free polyamines were therefore found to be differentially involved in the phenotypic plasticity of root architecture. A comparison of developmental effects and physiological concentrations suggested that agmatine and spermine in particular may play a significant role in the control of root development.     

Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs

The ability to map the position of ribosomes and their associated factors on mRNAs is critical for an understanding of translation mechanisms. Earlier approaches to monitoring these important cellular events characterized nucleotide sequences rendered nuclease-resistant by ribosome binding. While these approaches furthered our understanding of translation initiation and ribosome pausing, the pertinent techniques were technically challenging and not widely applied. Here we describe an alternative assay for determining the mRNA sites at which ribosomes or other factors are bound. This approach uses primer extension inhibition, or "toeprinting," to map the 3' boundaries of mRNA-associated complexes. This methodology, previously used to characterize initiation mechanisms in prokaryotic and eukaryotic systems, is used here to gain an understanding of two interesting translational regulatory phenomena in the fungi Neurospora crassa and Saccharomyces cerevisiae: (a) regulation of translation in response to arginine concentration by an evolutionarily conserved upstream open reading frame, and (b) atypical termination events that occur as a consequence of the presence of premature stop codons.     

Ly-49P activates NK-mediated lysis by recognizing H-2Dd

Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd alpha1/alpha2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.     

Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor

The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron-sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.     

Evaluation of mobilized peripheral blood CD34+ cells from patients with severe coronary artery disease as a source of endothelial progenitor cells

Background aimsThe distinction between hematopoietic stem cells (HSC) and endothelial progenitor cells (EPC) is poorly defined. Co-expression of CD34 antigen with vascular endothelial growth factor (VEGF) receptor (VEGFR2) is currently used to define EPC (1).MethodsWe evaluated the phenotypic and genomic characteristics of peripheral blood-derived CD34⁺ cells in 22 granulocyte–colony-stimulating factor (G-CSF)-mobilized patients with severe coronary artery disease and assessed the influence of cell selection and storage on CD34⁺ cell characteristics.ResultsThe median CD34⁺ cell contents in the products before and after enrichment with the Isolex 300i Magnetic Cell Selection System were 0.2% and 82.5%, respectively. Cell-cycle analysis showed that 80% of CD34⁺ cells were in G0 stage; 70% of the isolated CD34⁺ cells co-expressed CD133, a marker for more immature progenitors. However, less than 5% of the isolated CD34⁺ cells co-expressed the notch receptor Jagged-1 (CD339) and only 2% of the isolated CD34⁺ population were positive for VEGFR2 (CD309). Molecular assessment of the isolated CD34⁺ cells demonstrated extremely low expression of VEGFR2 and endothelial nitric oxide synthase (eNOS) and high expression of VEGF-A. Overnight storage at 4°C did not significantly affect CD34⁺ cell counts and viability. Storage in liquid nitrogen for 7 weeks did not affect the percentage of CD34⁺ cells but was associated with a 26% drop in cell viability.ConclusionsWe have demonstrated that the majority of isolated CD34⁺ cells consist of immature and quiescent cells that lack prototypic markers of EPC. High VEGF-A gene expression might be one of the mechanisms for CD34⁺ cell-induced angiogenesis.     

Heat stress proteins and myocardial protection: experimental model or potential clinical tool?

Heat stress proteins (hsp) are induced by a variety of stimuli including elevated temperature, ischaemia, hypoxia, pressure overload and some chemicals. They help to maintain the metabolic and structural integrity of the cell, as a protective response to external stresses. They are known to protect the myocardium from the damaging effects of ischaemia and reperfusion. The heat stress response results in accumulation of heat stress proteins. The beneficial effects associated with their expression include improved endothelial and mechanical recovery of the ischaemic heart. In addition, preservation of high energy phosphates and reduction in infarct size. It has also been shown that critical amounts of hsp70 are necessary to ensure protection of the myocardium. However, questions remain regarding the biochemical mechanisms underlying this protective effect. Alterations in the cell metabolism and chaperone function of cells expressing heat shock proteins, are thought to be responsible. Despite the obvious clinical benefits related to the heat stress response in a clinical setting, the application of this phenomena remains limited. Heat, both quantitatively and qualitatively is one of the best inducers of heat stress proteins. However, the effects of heat stress are nonspecific and intracellular damage is a common occurrence. The search for alternative stimuli, particularly within the fields of pharmacotherapy or genetic manipulation may offer more viable options, if the heat stress response is take its place as an established strategy for myocardial protection.     

Radiation induced esophageal adenocarcinoma in a woman previously treated for breast cancer and renal cell carcinoma

ABSTRACT: BACKGROUND: Secondary radiation-induced cancers are rare but well-documented as long-term side effects of radiation in large populations of breast cancer survivors. Multiple neoplasms are rare. We report a case of esophageal adenocarcinoma in a patient treated previously for breast cancer and clear cell carcinoma of the kidney CASE PRESENTATION: A 56 year-old non smoking woman, with no alcohol intake and no familial history of cancer; followed in the National Institute of Oncology of Rabat Morocco since 1999 for breast carcinoma, presented on consultation on January 2011 with dysphagia. Breast cancer was treated with modified radical mastectomy, 6 courses of chemotherapy based on CMF regimen and radiotherapy to breast, inner mammary chain and to pelvis as castration. Less than a year later, a renal right mass was discovered incidentally. Enlarged nephrectomy realized and showed renal cell carcinoma. A local and metastatic breast cancer recurrence occurred in 2007. Patient had 2 lines of chemotherapy and 2 lines of hormonotherapy with Letrozole and Tamoxifen assuring a stable disease. On January 2011, the patient presented dysphagia. Oesogastric endoscopy showed middle esophagus stenosing mass. Biopsy revealed adenocarcinoma. No evidence of metastasis was noticed on computed tomography and breast disease was controlled. Palliative brachytherapy to esophagus was delivered. Patient presented dysphagia due to progressive disease 4 months later. Jejunostomy was proposed but the patient refused any treatment. She died on July 2011 CONCLUSION: we present here a multiple neoplasm in a patient with no known family history of cancers. Esophageal carcinoma is most likely induced by radiation. However the presence of a third malignancy suggests the presence of genetic disorders.