Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.

The human CFTR protein expressed in CHO cells activates aquaporin-3 in a cAMP-dependent pathway: study by digital holographic microscopy.

The transmembrane water movements during cellular processes and their relationship to ionic channel activity remain largely unknown. As an example, in epithelial cells it was proposed that the movement of water could be directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) protein activity through a cAMP-stimulated aqueous pore, or be dependent on aquaporin. Here, we used digital holographic microscopy (DHM) an interferometric technique to quantify in situ the transmembrane water fluxes during the activity of the epithelial chloride channel, CFTR, measured by patch-clamp and iodide efflux techniques. We showed that the water transport measured by DHM is fully inhibited by the selective CFTR blocker CFTRinh172 and is absent in cells lacking CFTR. Of note, in cells expressing the mutated version of CFTR (F508del-CFTR), which mimics the most common genetic alteration encountered in cystic fibrosis, we also show that the water movement is profoundly altered but restored by pharmacological manipulation of F508del-CFTR-defective trafficking. Importantly, whereas activation of this endogenous water channel required a cAMP-dependent stimulation of CFTR, activation of CFTR or F508del-CFTR by two cAMP-independent CFTR activators, genistein and MPB91, failed to trigger water movements. Finally, using a specific small-interfering RNA against the endogenous aquaporin AQP3, the water transport accompanying CFTR activity decreased. We conclude that water fluxes accompanying CFTR activity are linked to AQP3 but not to a cAMP-stimulated aqueous pore in the CFTR protein.

A malaria merozoite surface protein (MSP1)-structure, processing and function

Merozoite surface protein-1 (MSP-1, also referred to as P195, PMMSA or MSA 1) is one of the most studied of all malaria proteins. The proteins. The protein is found in all malaria species investigated and structural studies on the gene indicate that parts of the molecule are well-conserved. Studies on Plasmodium falciparum have shown that the protein is in a processed form on the merozoite surface, a result of proteolytic cleavage of the large percursor molecule. Recent studies have identified some of these cleavage sites. During invasion of the new red cell most of the MSP1 molecule is shed from the parasite surface except for a small C-terminal fragment which can be detected in ring stages. Analysis of the structure of this fragment suggests that it contains two growth factor-like domains that may have a functional role.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Human IgG responses against the N-terminal region of Merozoite Surface Protein 1 of Plasmodium vivax

The complete primary structure of the gene encoding the Merozoite Surface Protein 1 of Plasmodium vivax (PvMSP-1) revealed the existence of interspecies conserved regions among the analogous proteins of other Plasmodia species. Here, three DNA recombinant clones expressing 50, 200 and 500 amino acids from the N-terminal region of the PvMSP-1 protein were used on ELISA and protein immunoblotting assays to look at the IgG antibody responses of malaria patients from the Brasilian amazon region of Rondônia. The results showed the existance of P. vivax and P. falciparum IgG antibodies directed against PvMSP-1 antigenic determinants expressed in the clones containing the first 200 and the following 500 amino acids of the molecule, but not within the one expressing the most N-terminal 50 amino acids. Interestingly, there was no correlation between the levels of these IgG antibodies and the previous number of malaria infections.

The Pf332 gene codes for a megadalton protein of Plasmodium falciparum asexual blood stages

We characterized the Plasmodium falciparum antigen 332 (Ag332) which is specifically expressed during the asexual intraerythrocytic cycle of the parasite. The corresponding Pf332 gene has been located in the subtelomeric region of chromosome 11. Furthermore, it is present in all strais so far analyzed and shows marked restriction length fragment polymorphism. Partial sequence and restriction endonuclease digestion of cloned fragments revealed that the Pf332 gene is composed of highly degenerated repeats rich is glutamic acid. Mung been nuclease digestion and Northern blot analysis suggested that Pf332 gene codes for a protein of about 700 kDa. These data were further confirmed by Western blot and immunoprecipitation of parasites extracts with an antiserum raised against a recombinant clone expressing part of the Ag332. Confocal immunofluorescence showed that Ag332 is translocated from the parasite to the surface of infected red blood cells within vesicle-like structures. In addition, Ag332 was detected on the surface of monkey erythrocytes infected with Plasmodium falciparum.

A recombinant hybrid protein as antigen for an anti-blood stage malaria vaccine: a study on the conservation of a protective component

Recently we have shown that two hybrid proteins expressed in Escherichia coli confer protective immunity to Aotus monkeys against an experimental Plasmodium falciparum infection (Knapp et al., 1992). Both hybrid proteins carry a sequence containing amino acids 631 to 764 of the serine stretch protein SERP (Knapp et al., 1989b). We have studied the diversity of this SERP region in field isolates of P. falciparum. Genomic DNA was extracted from the blood of six donors from different endemic areas of Brazil and West Africa. The SERP region encoding amino acids 630 to 781 was amplified by polymerase chain reaction (PCR) and sequenced. Only conserved amino acid substitutions in maximally two positions of the analyzed SERP fragment could be detected which supports the suitability of this SERP region as a component of anti-blood stage malaria vaccine.

Development of an immunoenzymatic assay using a monoclonal antibody against a 50-kDa catabolite from the P126 Plasmodium falciparum protein to the diagnosis of malaria infection

The WHO criterion of defering any donation of blood by a confirmed case of malaria for three years after cessation of therapy can not be applied in areas where malaria in endemic. For this reason we developed an immunoenzymatic assay for the detection of plasmodial antigens for blood screening in malararial endemic areas. So, we tested sera from 191 individuals. Among patients with active disease 100% of the cases of Plasmodium falciparum or mixed infections and 91.7% of those with P. vivax were positive for the presence of plasmodial antigens. The lower parasitaemia detected was 0.0003% for P. vivax malária. When the frequency of positive circulating malarial antigens was evaluated among asymptomatic and symptomatic individuals with negative TBS, positive results were found in respectively 38.7% and 17.7% of the individuals studied in the 30 days after confirmed malaria attack. Data provide by these assays have shown that ELISA seemed to be more sensitive than parasitological examination for malaria diagnosis. This test by virtue of its high sensivity and the facilities in processing a large number of specimens, can prove to be useful in endemic areas for the recognition of asymptomatic malaria and screening of blood donors.

T cell responses to repeat and non-repeat regions of the circumsporozoite protein detected in volunteers immunized with Plasmodium falciparum sporozoites

The design of malarial vaccine based on the circumsporozoite (CS) protein, a majuor surface antigen of the sporozoite stage of the malaria parasite, requires the identification of T and B cell epitopes for inclusion in recombinant or synthetic vaccine candidates. We have investigated the specificity and function of a series of T cell clones, derived from volunteers immunized with Plasmodium falciparum sporozoites in an effort to identify relevant epitopes in the immune response to the pre-erythrocytic stages of the parasite. CD4+ T cell clones were obtained wich specifically recognized a repetitive epitope located in the 5'repeat region of the CS protein. This epitope, when conjugated to the 3'repeat region in a synthetic MAPs construct, induced high titers of antisporozoite antibodies in C57B1 mice. A second T cell epitope, which mapped to aa 326-345 of the carboxy terminal, was recognized by lytic, as well as non-lytic, CD4+ T cells derived from the sporozoite-immunized volunteers. The demonstration of CD4+ CTL in the volunteers, and the recent studies inthe rodent model (Renia et al., 1991; Tsuji et al., 1990), suggested that CS-specific CD4+ T cells, in addition to their indirect role as helper cells in the induction of antibody and CD8 + effector cells, may also play a direct role in protection against sporozoite challenge by targeting EEF within the liver.

Distinct expression pattern of microtubule-associated protein-2 in human oligodendrogliomas and glial precursor cells.

Microtubule-associated protein 2 (MAP2), a protein linked to the neuronal cytoskeleton in the mature central nervous system (CNS), has recently been identified in glial precursors indicating a potential role during glial development. In the present study, we systematically analyzed the expression of MAP2 in a series of 237 human neuroepithelial tumors including paraffin-embedded specimens and tumor tissue microarrays from oligodendrogliomas, mixed gliomas, astrocytomas, glioblastomas, ependymomas, as well as dysembryoplastic neuroepithelial tumors (DNT), and central neurocytomas. In addition, MAP2-immunoreactive precursor cells were studied in the developing human brain. Three monoclonal antibodies generated against MAP2A-B or MAP2A-D isoforms were used. Variable immunoreactivity for MAP2 could be observed in all gliomas with the exception of ependymomas. Oligodendrogliomas exhibited a consistently strong and distinct pattern of expression characterized by perinuclear cytoplasmic staining without significant process labeling. Tumor cells with immunoreactive bi- or multi-polar processes were mostly encountered in astroglial neoplasms, whereas the small cell component in neurocytomas and DNT was not labeled. These features render MAP2 immunoreactivity a helpful diagnostic tool for the distinction of oligodendrogliomas and other neuroepithelial neoplasms. RT-PCR, Western blot analysis, and in situ hybridization confirmed the expression of MAP2A-C (including the novel MAP2+ 13 transcript) in both oligodendrogliomas and astrocytomas. Double fluorescent laser scanning microscopy showed that GFAP and MAP2 labeled different tumor cell populations. In embryonic human brains, MAP2-immunoreactive glial precursor cells were identified within the subventricular or intermediate zones. These precursors exhibit morphology closely resembling the immunolabeled neoplastic cells observed in glial tumors. Our findings demonstrate MAP2 expression in astrocytic and oligodendroglial neoplasms. The distinct pattern of immunoreactivity in oligodendrogliomas may be useful as a diagnostic tool. Since MAP2 expression occurs transiently in migrating immature glial cells, our findings are in line with an assumed origin of diffuse gliomas from glial precursors.

Plasmodium falciparum CS protein - prime malaria vaccine candidate: definition of the human CTL domain and analysis of its variation

Studies in mice have shown that immunity to malaria sporozoites is mediated primarily by citotoxic T lymphocytes (CTL) specific for epitopes within the circumsporozoite (CS) protein. Humans, had never been shown to generate CTL against any malaria or other parasite protein. The design of a sub-unit vaccine for humans ralies on the epitopes recognized by CTL being identified and polymorphisms therein being defined. We have developed a novel technique using an entire series of overlapping synthetic peptides to define the epitopes of the Plasmodium falciparum CS protein recognized by human CTL and have analyzed the sequence variation of the protein with respect to the identified CTL epitopic domain. We have demonstrated that some humans can indeed generate CTL. against the P. falciparum CS protein. Furthermore, the extent of variation observed for the CTL recognition domain is finite and the combination of peptides necessary for inclusion in a polyvalent vaccine may be small. If ways can be found to increase immune responsiveness, then a vaccine designed to stimulate CS protein-specific CTL activity may prevent malaria.

Neural Wiskott-Aldrich syndrome protein modulates Wnt signaling and is required for hair follicle cycling in mice.

The Rho family GTPases Cdc42 and Rac1 are critical regulators of the actin cytoskeleton and are essential for skin and hair function. Wiskott-Aldrich syndrome family proteins act downstream of these GTPases, controlling actin assembly and cytoskeletal reorganization, but their role in epithelial cells has not been characterized in vivo. Here, we used a conditional knockout approach to assess the role of neural Wiskott-Aldrich syndrome protein (N-WASP), the ubiquitously expressed Wiskott-Aldrich syndrome-like (WASL) protein, in mouse skin. We found that N-WASP deficiency in mouse skin led to severe alopecia, epidermal hyperproliferation, and ulceration, without obvious effects on epidermal differentiation and wound healing. Further analysis revealed that the observed alopecia was likely the result of a progressive and ultimately nearly complete block in hair follicle (HF) cycling by 5 months of age. N-WASP deficiency also led to abnormal proliferation of skin progenitor cells, resulting in their depletion over time. Furthermore, N-WASP deficiency in vitro and in vivo correlated with decreased GSK-3beta phosphorylation, decreased nuclear localization of beta-catenin in follicular keratinocytes, and decreased Wnt-dependent transcription. Our results indicate a critical role for N-WASP in skin function and HF cycling and identify a link between N-WASP and Wnt signaling. We therefore propose that N-WASP acts as a positive regulator of beta-catenin-dependent transcription, modulating differentiation of HF progenitor cells.

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays

Protein extracts obtained from male and female shistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3'end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generatingrelatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of proteins occured at the 5'end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide the F-10 HRE, evidence proteins having MWS of 30,45 and 65 kDNA. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Generation and phenotypic analysis of protein S-deficient mice.

Protein S (PS) is an important natural anticoagulant with potentially multiple biologic functions. To investigate further the role of PS in vivo, we generated Pros(+/-) heterozygous mice. In the null (-) allele, the Pros exons 3 to 7 have been excised through conditional gene targeting. Pros(+/-) mice did not present any signs of spontaneous thrombosis and had reduced PS plasma levels and activated protein C cofactor activity in plasma coagulation and thrombin generation assays. Tissue factor pathway inhibitor cofactor activity of PS could not be demonstrated. Heterozygous Pros(+/-) mice exhibited a notable thrombotic phenotype in vivo when challenged in a tissue factor-induced thromboembolism model. No viable Pros(-/-) mice were obtained through mating of Pros(+/-) parents. Most E17.5 Pros(-/-) embryos were found dead with severe intracranial hemorrhages and most likely presented consumptive coagulopathy, as demonstrated by intravascular and interstitial fibrin deposition and an increased number of megakaryocytes in the liver, suggesting peripheral thrombocytopenia. A few E17.5 Pros(-/-) embryos had less severe phenotype, indicating that life-threatening manifestations might occur between E17.5 and the full term. Thus, similar to human phenotypes, mild heterozygous PS deficiency in mice was associated with a thrombotic phenotype, whereas total homozygous deficiency in PS was incompatible with life.

Immunoelectron microscopical identification of the uncoupling protein in brown adipose tissue mitochondria.

The distribution of the uncoupling protein (UCP) in brown adipocyte mitochondria of the hibernant Muscardinus avellanarius was obtained by ultrastructural immunocytochemistry. In both cryosections and sections of Lowicryl-embedded material UCP was localized in the mitochondrial cristae of brown adipocytes, but not in liver mitochondria. It should now be possible to easily identify the morphology of cells committed to BAT differentiation in the tissue as well as in cell culture.