High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.     

Pyogenic granuloma: an unrecognized cause of gastrointestinal bleeding

Pyogenic granuloma is a lobular capillary hemangioma that mostly occurs on the skin, but it is also encountered on the mucosal surface of the oral cavity. Only a few cases in other parts of the digestive tract have been reported in Japanese patients. In this report, two Caucasian patients are described, who presented with gastrointestinal bleeding due to the presence of a pyogenic granuloma. One was located in the distal esophagus and could be treated with local excision and laser-photocoagulation therapy. The other one was located in the small intestine and was removed by surgical resection. Although extremely rare, pyogenic granuloma as a cause of gastrointestinal bleeding needs consideration. The lesion is benign, presumably reactive and can be adequately treated by excision or laser photocoagulation. Immunohistochemistry and/or polymerase chain reaction for herpesvirus 8 can reliably distinguish pyogenic granuloma from Kaposis sarcoma, an important differential diagnosis.     

Cellular and molecular analyses of major histocompatibility complex (MHC) restricted and non-MHC-restricted effector cells recognizing renal cell carcinomas: problems and perspectives for immunotherapy

 Renal cell carcinomas belong to the small group of tumors that are able to induce antitumor responses. Here we describe two general types of cytotoxic effector lymphocytes that can eliminate autologous tumor cells and discuss the role that major histocompatibility complex encoded molecules play in governing their specificities. Improved understanding of the cellular and molecular basis of renal cell carcinoma recognition opens new avenues of research with the potential to develop better immunotherapies for patients with metastatic disease. Received: 24 July 1996 / Accepted: 1 November 1996     

The anti-apoptotic factor Bcl-2 can functionally substitute for the B cell survival but not for the marginal zone B cell differentiation activity of BAFF

The TNF family ligand B cell-activating factor (BAFF, BLyS, TALL-1) is an essential factor for B cell development. BAFF binds to three receptors, BAFF-R, transmembrane activator and CAML interactor (TACI), and B cell maturation antigen (BCMA), but only BAFF-R is required for successful survival and maturation of splenic B cells. To test whether the effect of BAFF is due to the up-regulation of anti-apoptotic factors, TACI-Ig-transgenic mice, in which BAFF function is inhibited, were crossed with transgenic mice expressing FLICE-inhibitory protein (FLIP) or Bcl-2 in the B cell compartment. FLIP expression did not rescue B cells, while enforced Bcl-2 expression restored peripheral B cells and the ability to mount T-dependent antibody responses. However, many B cells retained immaturity markers and failed to express normal amounts of CD21. Marginal zone B cells were not restored and the T-independent IgG3, but not IgM, response was impaired in the TACI-IgxBcl-2 mice. These results suggest that BAFF is required not only to inhibit apoptosis of maturating B cells, but also to promote differentiation events, in particular those leading to the generation of marginal zone B cells.     

Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay

Mutations of the p53 gene are the most frequently observed genetic changes in human cancers; often leading to an overexpression of the wild-type (wt) p53 protein. Demonstrable T cell reactivity against tumor cells overexpressing wt or mutant p53-derived peptides could support the application of such epitopes in cancer immunotherapies. As the binding of peptide to MHC class I molecules is a prerequisite for antigen-specific T cell recognition, we evaluated the ability of wt and mutant p53 peptides to bind to HLA-A2.1 using two independent flow cytometry-based assay systems, the T2 major histocompatibility complex (MHC) class I peptide stabilization assay (stabilization assay) and the peptide-induced MHC class I reconstitution assay (reconstitution assay). The twenty selected wt sequences each conformed to the previously reported HLA-A2.1 peptide binding motif. Seven of the wt p53 and 2/13 mutant p53 peptides derived from the previously chosen wt peptides bound to HLA-A2.1 in both the stabilization and the reconstitution assays. An additional six wt and six mutant p53 peptides, presumably exhibiting lower affinity for HLA-A2.1, were identified only in the reconstitution assay. Those p53 peptides binding HLA-A2.1 may provide useful immunogens for the generation of HLA-A2.1-restricted cytolytic T lymphocytes in vitro and in vivo.     

Effects of sodium and GTP on the binding kinetics of [3H]diprenorphine in NG 108-15 cell membranes

Summary Equilibrium binding isotherms of [³H]diprenorphine in membranes from NG 108-15 cells are consistent with a homogenous population of binding sites. Upon addition of Na⁺, Mg²⁺ and GTP, only a 2-fold reduction in affinity with a minor decrease in the number of sites is observed. Dissociation curves of [³H]diprenorphine, however, are clearly biphasic: in the absence of Na⁺, Mg²⁺ and GTP, 80% of the bound ligand dissociates slowly with at _1/2 of 100 min, and only 20% rapidly (t _1/2 4.5 min). In the presence of Mg²⁺, nearly all the binding is found in the slowly dissociating form. Upon the addition of either Na⁺ or GTP, 20–30% of the binding dissociates more rapidly. The rate constant of the rapidly dissociating form generated by Na⁺, however, is 2.5 times greater than that induced by the presence of GTP. Thus, the addition of both, Na⁺ and GTP, converts about 80% of the receptor into a very fast dissociating form (t _1/2 1.7 min).Exposure of intact cells to pertussis toxin (10 ng/ml) or treatment of membranes with N-ethyl maleimide (500 M), strongly reduces the proportion of the slowly dissociating component. Following these treatments, the effect of GTP is reduced or abolished, but that of Na⁺ remains unaffected.We conclude from these data that the effects of Na⁺ and GTP are not only distinct in site but also in mechanism of action and that there are three forms of opioid receptors that can be differentiated by their kinetic properties. The slowly dissociating receptor form requires a functional N unit.