Characterization of aminopeptidase N from Torpedo marmorata kidney

Summary— A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C₁₂E₉), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm² of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.     

Non-photochemical fluorescence quenching and the diadinoxanthin cycle in a marine diatom

The diadinoxanthin cycle (DD-cycle) in chromophyte algae involves the interconversion of two carotenoids, diadinoxanthin (DD) and diatoxanthin (DT). We investigated the kinetics of light-induced DD-cycling in the marine diatom Phaeodactylum tricornutum and its role in dissipating excess excitation energy in PS II. Within 15 min following an increase in irradiance, DT increased and was accompanied by a stoichiometric decrease in DD. This reaction was completely blocked by dithiothreitol (DTT). A second, time-dependent, increase in DT was detected 20 min after the light shift without a concomitant decrease in DD. DT accumulation from both processes was correlated with increases in non-photochemical quenching of chlorophyll fluorescence. Stern-Volmer analyses suggests that changes in non-photochemical quenching resulted from changes in thermal dissipation in the PS II antenna and in the reaction center. The increase in non-photochemical quenching was correlated with a small decrease in the effective absorption cross section of PS II. Model calculations suggest however that the changes in cross section are not sufficiently large to significantly reduce multiple excitation of the reaction center within the turnover time of steady-state photosynthetic electron transport at light saturation. In DTT poisoned cells, the change in non-photochemical quenching appears to result from energy dissipation in the reaction center and was associated with decreased photochemical efficiency. D1 protein degradation was slightly higher in samples poisoned with DTT than in control samples. These results suggest that while DD-cycling may dynamically alter the photosynthesis-irradiance response curve, it offers limited protection against photodamage of PS II reaction centers at irradiance levels sufficient to saturate steady-state photosynthesis.Abbreviations CAP chloramphenicol - D1 PS II reaction center protein - DD diadinoxanthin - DD cycle-diadinoxanthin cycle - DT diatoxanthin - DTT dithiothreitol - FCP fucoxanthin chlorophyll a-c protein - F_m maximum fluorescence yield in the dark-adapted state - F_o minimum fluorescence yield in the dark-adapted state - F_m and F_o maximum and minimum fluorescence yields respectively in some light adapted state - F_v maximum variable fluorescence yield in the dark-adapted state - I_k Irradiance at the intercept of the initial slope of the photosynthesis-irradiance curve and the maximum photosynthetic rate - k_D first order rate constant for nonradiative de-excitation of excitions in the PS II antenna - k_d first order rate constant for non-radiative de-excitation of excitons in the PS II reaction center - k_F first order rate constant for fluorescence - k_T first order rate constant for exciton transfer to the reaction center - k_t first order rate constant for exciton transfer from the reaction center to the antenna - Rubisco ribulose bisphosphate carboxylase - SV_m Stern-Volmer quenching coefficient of the maximum fluorescence yield - SV_o Stern-Volmer quenching coefficient of the miniximum fluorescence yield - _{PS II} apparent absorption cross-section of PS II - _arr average interval between exciton arrival to the PS II reaction center (ms) - _rem average interval between electron turnover during photosynthesis in the PS II reaction center (ms) - _d the probability that an exciton is non-radiatively dissipated in the reaction center - _T the probability that an exciton in the antenna is transferred to the reaction center - _t the probability that an exciton is transferred back from the reaction center to the antenna     

Expression of the hepatitis delta virus large and small antigens in transgenic mice.

Simultaneous infection with hepatitis delta virus (HDV) and hepatitis B virus (HBV) in humans is often associated with severe viral liver disease including fulminant hepatitis. Since HBV is thought to be noncytopathic to the hepatocyte, the enhanced disease severity observed during dual infection has been attributed to either simultaneous immune responses against the two viruses or direct cytotoxic effects of HDV products on the hepatocyte or both. To examine these alternate possibilities, we produced transgenic mice that express the small and large delta antigens (HDAg) in hepatocyte nuclei at levels equal to those observed during natural HDV infection. No biological or histopathological evidence of liver disease was detectable during 18 months of observation, suggesting that neither the large nor small form of HDAg is directly cytopathic to the hepatocyte in vivo.     

In vivo replication of the hamster polyomavirus genome and generation of specific deletions in the process of lymphomagenesis.

Hamster polyomavirus (HaPV) causes lymphomas when injected into newborn hamsters. These tumors are virus-free but accumulate large amounts of deleted extrachromosomal viral genomes. In order to identify the major sites of virus replication in animals, we have monitored the HaPV DNA present in different organs at various times after injection. The data demonstrate that viral replication preferentially occurs in lymphoid organs. Lymphoma-associated viral genomes display specific deletions. PCR analysis shows that such viral genomes are the only variants detectable in infected animals, suggesting that they are generated by a specific cellular mechanism. We have tested the possible role of the lymphoid cell-specific V(D)J recombination activity in the generation of these specific variants. Our results indicate that this mechanism is not solely responsible for the viral genome rearrangement, if involved at all.     

Chromosome 4q35 haplotypes and DNA rearrangements segregating in affected subjects of 19 Italian families with facioscapulohumeral musculatur dystrophy (FSHD)

Four DNA markers on the distal long arm of chromosome 4 have been analyzed for their linkage to facioscapulohumeral muscular dystrophy locus (FSHD) in a series of 16 Italian families. We found that, in two families, the disease is not linked to the 4q35 markers, indicating the presence of genetic heterogeneity among Italian FSHD families. Linkage analysis in the remaining families supports the order cen-D4S171-D4S163-D4S139-D4S810-FSHD-qter, in agreement with the physical map from the literature. EcoRI digestion and hybridization with the distal marker p13E-11 (D4S810) detected DNA rearrangements in the affected members of both sporadic and familial cases of FSHD, with family-specific fragments ranging in size between 15 kb and 28 kb. In three sporadic FSHD cases, the appearance of a new small fragment not present in either parent was clearly associated with the development of FSHD disease. However, in the familial cases analyzed, we observed two recombinations between all four 4q35 markers and the disease locus in apparently normal subjects, leaving open the possibility of nonpenetrance of the FSHD mutation.

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Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.