Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein

Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.     

Presence and release of SR-17 (chromogranin B(586-602)) in the porcine splenic nerve and its enzymatic degradation by CD26/dipeptidyl peptidase IV

Using the pig splenic nerve as a model, we investigated the proteolytic processing of porcine chromogranin B (CgB) during its axonal transport. An ELISA was developed for SR-17 (CgB(586-602)), a novel CgB-derived peptide, originally found in the adrenal medulla. The results demonstrate that CgB is processed in an early stage during its axonal transport. Immunohistochemical data, based on a rabbit anti-SR-17 antiserum, show that the spleen CgB/SR-17 is exclusively present in the nerve endings. No SR-17 immunoreactivity (IR) was found in splenocytes. We also provide evidence that SR-17 is co-released with noradrenaline (NA) upon electrical stimulation of the splenic nerve. Its release is frequency-dependent and strongly enhanced in the presence of the alpha-blocking agent phentolamine. In addition, we show that the new CgB-peptide can serve as a substrate for the lymphocyte surface glycoprotein CD26, also known as dipeptidyl peptidase IV (DPP IV), generating a new peptide ER-15 (CgB(588-602)).     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3)

Secretion of cytolytic granules content at the immunological synapse is a highly regulated process essential for lymphocyte cytotoxicity. This process requires the rapid transfer of perforin containing lytic granules to the target cell interface, followed by their docking and fusion with the plasma membrane. Defective cytotoxicity characterizes a genetically heterogeneous condition named familial hemophagocytic lymphohistiocytosis (FHL), which can be associated with perforin deficiency. The locus of a perforin (+) FHL subtype (FHL3), observed in 10 patients, was mapped to 17q25. This region contains hMunc13-4, a member of the Munc13 family of proteins involved in vesicle priming function. HMunc13-4 mutations were shown to cause FHL3. HMunc13-4 deficiency results in defective cytolytic granule exocytosis, despite polarization of the secretory granules and docking with the plasma membrane. Expressed tagged hMunc13-4 localizes with cytotoxic granules at the immunological synapse. HMunc13-4 is therefore essential for the priming step of cytolytic granules secretion preceding vesicle membrane fusion.     

Evolution of the proto-MHC ancestral region: more evidence for the plesiomorphic organisation of human chromosome 9q34 region

The present day structure of the vertebrate major histocompatibility complex (MHC) and its three paralogous regions has always been a focus of interest. In a recent study, nine human anchor genes located in the MHC region were cloned from a Branchiostoma floridae (amphioxus) cosmid library. The identification and analysis of 31 surrounding genes led to the most probable model of two rounds of en bloc duplication giving rise to these regions. These events were estimated to have occurred after the cephalochordata-craniata divergence [approximately 766 million years ago (Mya)] and before the Gnathostomata radiation (approximately 528 Mya). Furthermore, it was also shown that after this large-scale duplication one of these regions, corresponding to the human 9q33-q34, had retained an ancestral organisation. In the present study, four new cosmids in the amphioxus proto-MHC region were identified by the chromosomal walking technique. These cosmids were sequenced, and their structural annotation was performed, leading to the prediction of eleven genes. Their phylogenetic relationships among species corroborate the results obtained previously and provide more evidence for the plesiomorphic state of the human chromosome 9q33-34 MHC paralogous region.An erratum to this article can be found at     

Relationship between haemolytic and sphingomyelinase activities in a partially purified beta-like toxin from Staphylococcus schleiferi

beta-Toxins of staphylococcal species possess dual activity in that they can both lyse erythrocytes (by 'hot-cold' lysis) and catalyse hydrolysis of membrane-associated sphingomyelin. However, the precise relationship between these two activities has not been extensively studied. We have partially purified a beta-like toxin from culture supernatants of Staphylococcus schleiferi N860375 which exhibits both 'hot-cold' lysis of erythrocytes and neutral sphingomyelinase activities. This toxin has a strong preference for sheep erythrocytes, the membranes of which are rich in sphingomyelin. Kinetic analysis suggests that haemolysis and sphingomyelinase activities are very closely associated obeying identical Michaelis-Menten kinetics. However, pre-treatment with antibodies to Staphylococcus aureus beta-toxin, Ca(2+), dithiothreitol and phenylmethylsulfonyl fluoride appear to inhibit sphingomyelinase activity significantly more strongly than haemolysis while Mg(2+) activates sphingomyelinase activity more strongly than haemolysis. We attribute these effects to differences in binding properties in the two assays. Micropurification by both sphingosylphosphocholine-agarose affinity chromatography and preparative electrophoresis revealed that the 34-kDa toxin associates non-covalently with individual proteins.     

Induction of cytolytic T lymphocytes by immunization of mice with an adenovirus containing a mouse homolog of the human MAGE-A genes

The genes of the MAGE-A family code for antigens that are strictly tumor-specific and are shared by many human tumors. Melanoma patients have been immunized against these antigens and some tumor regressions have been observed. However, no unequivocal evidence of cytolytic T cell responses has been obtained by analyzing the blood lymphocytes of these patients. Hence it was considered worthwhile to examine in mouse systems whether or not immunization against antigens derived from the mouse Mage homologs can produce cytolytic T cell responses. We have identified an antigenic peptide encoded by mouse gene Mage-a2, and here we show that immunization of DBA/2 mice with a recombinant adenovirus containing either just the sequence encoding this peptide or a large part of the Mage-a2 coding sequence produces strong cytolytic T cell responses. The Mage-a2 system should prove useful for the comparison of vaccination modalities that could be applied to human patients in therapeutic vaccination trials with MAGE antigens. Received: 1 June 2000 / Accepted: 17 August 2000     

Enhanced Virus Clearance by Early Inducible Lymphocytic Choriomeningitis Virus-Neutralizing Antibodies in Immunoglobulin-Transgenic Mice

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin μ heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host’s capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin μ heavy and the κ light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.Against many cytopathic viruses such as poliovirus, influenza virus, rabies virus, and vesicular stomatitis virus, protective virus-neutralizing antibodies are generated early, within 1 week after infection (3, 31, 36, 44, 49). In contrast, several noncytopathic viruses (e.g., human immunodeficiency virus and hepatitis viruses B and C in humans or lymphocytic choriomeningitis virus [LCMV] in mice) elicit poor and delayed virus-neutralizing antibody responses (1, 7, 20, 24, 27, 35, 45, 48).In the mouse, the natural host of LCMV, the acute LCMV infection is predominantly controlled by cytotoxic T lymphocytes (CTLs) in an obligatory perforin-dependent manner (13, 18, 28, 50). In addition to the CTL response, LCMV-specific antibodies are generated. Early after infection (by day 8), a strong antibody response specific for the internal viral nucleoprotein (NP) is mounted (7, 19, 23, 28). These early LCMV NP-specific antibodies exhibit no virus-neutralizing capacity (7, 10). Results from studies of B-cell-depleted mice and B-cell-deficient mice implied that the early LCMV NP-specific antibodies are not involved in the clearance of LCMV (8, 11, 12, 40). Late after infection (between days 30 and day 60), LCMV-neutralizing antibodies develop (7, 19, 22, 28, 33); these antibodies are directed against the surface glycoprotein (GP) of LCMV (9, 10). LCMV-neutralizing antibodies have an important function in protection against reinfection (4, 6, 38, 41, 47).In some viral infections, subprotective virus-neutralizing antibody titers can enhance disease rather than promote host recovery (i.e., exhibit antibody-dependent enhancement of disease [ADE] [14, 15, 21, 46]). For example, neutralizing antibodies are involved in the resolution of a primary dengue virus infection and in the protection against reinfection. However, if subprotective neutralizing antibody titers are present at the time of reinfection, a severe form of the disease (dengue hemorrhagic fever/dengue shock syndrome [15, 21]), which might be caused by Fc receptor-mediated uptake of virus-antibody complexes leading to an enhanced infection of monocytes (15, 16, 25, 39), can develop. Similarly, an enhancement of disease after intracerebral (i.c.) LCMV infection was observed in mice which had been treated with virus-neutralizing antibodies before the virus challenge (6). ADE in LCMV-infected mice was either due to an enhanced infection of monocytes by Fc receptor-mediated uptake of antibody-virus complexes or due to CTL-mediated immunopathology caused by an imbalanced virus spread and CTL response.To analyze whether LCMV-neutralizing antibodies generated early after infection improve the host’s capacity to clear the virus or enhance immunopathological disease, immunoglobulin (Ig)-transgenic mice expressing LCMV-neutralizing IgM antibodies were generated. After LCMV infection of transgenic mice expressing the Ig heavy chain (H25 transgenic mice), LCMV-neutralizing serum antibodies were mounted within 8 days, which significantly improved the host’s capacity to eliminate LCMV. H25 transgenic mice did not show any signs of ADE after i.c. LCMV infection.Transgenic mice expressing the Ig heavy and light chains (HL25 transgenic mice) exhibited spontaneous LCMV-neutralizing serum antibodies and confirmed the protective role of preexisting LCMV-neutralizing antibodies, even though the neutralizing serum antibodies were of the IgM isotype. Similar to mice which had been treated with LCMV-neutralizing antibodies, HL25 transgenic mice developed an enhanced disease after i.c. LCMV infection, which indicated that ADE was due to an imbalance between virus spread and CTL response. Thus, the early-inducible LCMV-neutralizing antibody response significantly enhanced clearance of the acute infection without any risk of causing ADE.