"Cold-sensitive" mutants of the Lac repressor

Thirteen of more than 4,000 single-amino-acid-replacement mutants of the Lac repressor, generated by suppression of amber nonsense mutants, were characterized as having a cold-sensitive phenotype. However, when expressed as missense mutations, none of the replacements cause cold sensitivity, implicating the suppression mechanism as being responsible for this phenotype.     

Functional and intracellular signaling differences associated with the Helicobacter pylori AlpAB adhesin from Western and East Asian strains

Following adhesion of Helicobacter pylori to gastric epithelial cells, intracellular signaling leads to cytokine production, which causes H. pylori-related gastric injury. Two adjacent homologous genes (alpA and alpB), which encode H. pylori outer membrane proteins, are thought to be associated with adhesion and cytokine induction. We co-cultured gastric epithelial cells with wild type H. pylori strains and their corresponding alpA/alpB-deleted mutants (DeltaalpAB). Results were confirmed by complementation. Flow cytometry confirmed that AlpAB was involved in cellular adhesion. Deletion of alpAB reduced interleukin (IL)-6 induction in gastric epithelial cells. Deletion of alpAB reduced IL-8 induction with East Asian but not with Western strains. All AlpAB-positive strains tested activated the extracellular signal-regulated kinase, c-Fos, and cAMP-responsive element-binding protein. Activation of the Jun-N-terminal kinase, c-Jun, and NF-kappaB was exclusive to AlpAB from East Asian strains. DeltaalpAB mutants poorly colonized the stomachs of C57BL/6 mice and were associated with lower mucosal levels of KC and IL-6. Our results suggest that AlpAB may induce gastric injury by mediating adherence to gastric epithelial cells and by modulating proinflammatory intracellular signaling cascades. Known geographical differences in H. pylori-related clinical outcomes may relate to differential effects of East Asian and Western types of AlpAB on NF-kappaB-related proinflammatory signaling pathways.     

Recoverin as a cancer-retina antigen

In photoreceptor cells the Ca²⁺-binding protein recoverin controls phosphorylation of the visual receptor rhodopsin by inhibiting rhodopsin kinase (GRK-1). It can also serve as a paraneoplastic antigen in the development of retinal degeneration in some patients with cancer. The aberrant expression of recoverin in cancer cells and the presence of autoantibodies against recoverin are essential for the occurrence of cancer-associated retinopathy, which finally results in the apoptosis of photoreceptor cells. Noteworthy in cancer patients, the aberrant recoverin expression and the appearance of autoantibodies against recoverin are more frequent than paraneoplastic syndromes. We suggest the term “cancer-retina antigens” for this kind of proteins like recoverin that are solely expressed in retina and tumor tissues and evoke antibodies and/or T cells in patients with cancer. The rare development of a paraneoplastic syndrome is possibly caused by this immune response and probably depends on further events allowing to overcome the blood–retina barrier and the immune privileged status of the retina. It is still unknown whether aberrantly expressed recoverin could have a specific function in cancer cells, though it is suggested that it can be functionally associated with G-protein-coupled receptor kinases. This paper reviews the present knowledge on paraneoplastic syndromes associated with the aberrant expression of recoverin. A possible application of recoverin as a potential target for immunotherapy of cancer is discussed.This article is a symposium paper from the conference “Progress in Vaccination against Cancer 2005 (PIVAC 5)”, held in Athens, Greece, on 20–21 September 2005.     

Immunotherapy of renal cell carcinoma

Carcinomas of the kidney generally have a poor prognosis and respond minimally to classical radiotherapy or chemotherapy. Immunotherapy constitutes an interesting alternative to these established forms of treatment, and indeed, cytokine-based therapies have been used for many years, leading to favorable clinical responses in a small subset of patients. During the past few years, immunotherapeutical trials targeting renal cell tumor-associated antigens have also been reported, with diverse passive or active approaches using antibodies or aimed at activating tumor-directed T lymphocytes. The following review presents the results and the progress made in the field, including classical cytokine treatments, non-myeloablative stem cell transplantation and antigen specific-based trials, with special focus on T-cell studies. In consideration of the few specific molecular targets described so far for this tumor entity, current strategies which can lead to the identification of new relevant antigens will be discussed. Hopefully these will very soon contribute to an improvement in renal cell carcinoma specific immunotherapy and its evaluation.     

Assessment of CD4<Superscript>+</Superscript> T cells specific for the tumor antigen SSX-1 in cancer-free individuals

Proteins encoded by genes of the SSX family are specifically expressed in tumors and are therefore relevant targets for cancer immunotherapy. One of the first identified family members, SSX-1, is expressed in a large fraction of synovial sarcomas as a fusion protein together with the product of the SYT gene. In addition, the full-length SSX-1 antigen is frequently expressed in tumors of several other histological types such as sarcoma, melanoma, hepatocellular carcinoma, ovarian cancer and myeloma. To date, however, SSX-1 specific T cell responses have not been investigated and no SSX-1 derived T cell epitopes have been described. Here, we have assessed the presence of CD4(+) T cells directed against the SSX-1 antigen in circulating lymphocytes of cancer-free individuals. After a single in vitro stimulation with a pool of peptides spanning the entire SSX-1 protein we could detect and isolate SSX-1-specific CD4(+) T cells from 5/5 donors analyzed. SSX-1-specific polyclonal populations isolated from these cultures recognized peptides located in three distinct regions of the protein containing clusters of sequences with significant predicted binding to frequently expressed MHC class II alleles. Characterization of specific clonal CD4(+) T cell populations derived from one donor allowed the identification of several naturally processed epitopes recognized in association with HLA-DR. These data document the existence of a significant repertoire of CD4(+) T cells specific for SSX-1 derived sequences in circulating lymphocytes of any individual that can be exploited for the development of both passive and active immunotherapeutic approaches to control disease evolution in cancer patients.     

Spitting out information: Trigona bees deposit saliva to signal resource locations

Stingless bees of the species Trigona spinipes (Fabricius 1793) use their saliva to lay scent trails communicating the location of profitable food sources. Extracts of the cephalic labial glands of the salivary system (not the mandibular glands, however) contain a large amount (approx. 74%) of octyl octanoate. This ester is also found on the scent-marked substrates at the feeding site. We demonstrate octyl octanoate to be a single compound pheromone which induces full trail following behaviour. The identification of the trail pheromone in this widely distributed bee makes it an ideal organism for studying the mechanism of trail following in a day flying insect.     

Structure-activity based study of the Smac-binding pocket within the BIR3 domain of XIAP

A small series of peptide mimics was designed and synthesized to contain a heterocyclic ring in place of the potentially labile N-terminal peptide bond of the tetrapeptide containing the Smac-XIAP-binding motif. Two Smac mimics were shown to bind to the BIR3 domain of XIAP with moderate affinity and one displayed increased activity in cells relative to the Smac peptides. The structures of BIR3-XIAP in complex with a Smac peptide and a peptide mimic were solved and analyzed to elucidate the structure-activity relationship surrounding the Smac-binding domain within BIR3-XIAP.