Prevention of murine cryoglobulinemia and associated pathology by monoclonal anti-idiotypic antibody

A murine IgG3 mAb, clone 6-19, derived from non-manipulated autoimmune MRL/MpJ-lpr/lpr mice is a rheumatoid factor specific for IgG2a and is able to generate cryoglobulins via nonspecific IgG3 Fc-Fc interaction. Intraperitoneal passive transfer of ascites containing the 6-19 mAb into BALB/c mice induces, within 18 h, remarkable pathology characterized by skin vasculitis and acute glomerulonephritis associated with cryoglobulinemia. In order to evaluate the possibility of modulating the development of tissue lesions by an anti-Id antibody, we have raised an IgG2b anti-Id mAb specific to the 6-19 mAb. The cryoprecipitation of 6-19 mAb was completely inhibited in the presence of excess amounts of anti-Id mAb in vitro. In vivo, pretreatment of BALB/c mice with anti-6-19 anti-Id mAb inhibited development of skin vasculitis and glomerulonephritis induced by the 6-19 mAb. The cryoglobulin formation was markedly diminished due to enhanced elimination of the 6-19 mAb from the circulation. In contrast, pretreatment with an IgM anti-IgG3 rheumatoid factor mAb neither protected nor aggravated the development of tissue lesions. These results suggest possible implications in the anti-Id treatment of similar vascular diseases in man.     

IgG3 is the major source of cryoglobulins in mice

A total of 20 of 23 IgG3 mAb derived from unmanipulated autoimmune MRL/MpJ-lpr/lpr mice was shown to generate cryoglobulins which were composed exclusively of IgG3. Although three IgG3 mAb failed to develop cryoglobulins, they were able to bind nonspecifically to any IgG3 molecules as efficiently as cryoprecipitable IgG did. The direct role of the gamma 3 constant region for the generation of cryoglobulins was demonstrated by the following findings: 1) the cryoglobulin activity was independent of the specificity of the IgG3 mAb, 2) no mAb other than those of the IgG3 subclass, including IgM rheumatoid factors (RF), generated cryoglobulins, and 3) the cryoglobulin activity was gained after the Ig class switch of mAb from IgM to IgG3. Analysis of Ig components in three different sources of cryoglobulins, either induced by the injection of bacterial LPS or by the infection with Plasmodium yoelii in BALB/c mice or developed spontaneously in MRL/MpJ-lpr/lpr mice, revealed the selective concentration of IgG3 in these cryoglobulins; greater than 99%, 73% and 58% of IgG recoverable from these three cryoglobulins, respectively, were IgG3. This further attests to the major role of IgG3 in the generation of cryoglobulins in mice. In addition, the enhanced formation and even induction of IgG3 cryoglobulins in the presence of IgM anti-IgG3 RF mAb, and the enrichment of IgM RF in LPS- or malaria-induced cryoglobulins indicated that IgM RF can be involved in the generation of cryoglobulins by interacting with noncryoprecipitable IgG3 as well as cryoprecipitable IgG3.     

Mutations induced by benzo[a]pyrene diolepoxide at the hprt locus in human T-lymphocytes in vitro

Human T-lymphocytes have been treated with benzo[a]pyrene diolepoxide (BPDE) in vitro and T-cell clones mutated in the hprt gene have been isolated. The mutant frequencies in BPDE-treated T-cell cultures were on average 24-fold higher than those of untreated cultures. Thus, BPDE is a potent inducer of gene mutation in this system. In order to examine which types of mutations are induced by BPDE in human cells, 41 spontaneous and 44 BPDE-induced mutant clones have been characterized using the Southern blot technique. In addition, rearrangements of the T-cell-receptor beta and gamma loci have been used to determine the proportion of isolated clones that are unique, and thus likely to represent independent mutational events. Out of 23 independent spontaneous mutants 4 had large hprt alterations that could be detected on Southern blots. Two of these alterations, deletions of exons 2-6, have been confirmed using PCR of hprt cDNA and direct sequencing of the PCR product. All 33 independent BPDE-induced mutants had normal hprt restriction patterns which indicates that BPDE is mainly a point mutagen in this system.     

Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region.

In-frame and frameshift mutations were introduced into the pre-S region (1,212 base pairs) of duck hepatitis B virus. The in-frame mutants retained the inserted 12 nucleotides, while the frameshift mutants either reverted to wild type or exhibited a 10-nucleotide compensatory deletion downstream of the original mutation site. Thus, although duck hepatitis B virus has a compact and highly economical genome organization, it can replicate despite alterations of up to 9 amino acid codons in the pre-S and P open reading frames.     

Effects of MPTP, MPP+ and paraquat on mitochondrial potential and oxidative stress

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+) and 1,1-dimethyl-4,4-bipyridinium (paraquat) upon the electrical potential across the plasma and mitochondrial membranes within synaptosomes has been investigated. MPTP selectively depressed plasma membrane potential while MPP+ specifically reduced mitochondrial potential. The structurally similar compound paraquat had no effect on either membrane potential. Enhancement of the lipid peroxidative activity with an Fe-ADP complex depressed both potentials. Paraquat effected increased peroxidative activity in brain homogenates that was less pronounced than that due to Fe-ADP. MPTP reduced basal but stimulated Fe-ADP enhanced peroxidation. The mechanisms underlying the toxicity of MPP+ are likely to differ from those of paraquat, primarily involving impaired mitochondrial function rather than increased oxidative stress.     

Metabolism of Pyrene by the Basidiomycete Crinipellis stipitaria and Identification of Pyrenequinones and Their Hydroxylated Precursors in Strain JK375

The metabolism of pyrene, a polycyclic aromatic hydrocarbon, by submerged cultures of the basidiomycete Crinipellis stipitaria was studied. After incubation for 68 h at 25°C in a 20-liter fermentor with complex medium and 20 mg of pyrene per liter, five metabolites were detected. The compounds were isolated by preparative high-performance liquid chromatography on RP18 and DIOL gels. By UV, infrared, and ¹H nuclear magnetic resonance spectroscopy and mass spectrometry, 1-hydroxypyrene, 1,6-dihydroxypyrene, 1,8-dihydroxypyrene, 1,6-pyrenequinone, and 1,8-pyrenequinone were identified. 1,6- and 1,8-dihydroxypyrene were obtained from fungal cultures for the first time. The formation of these metabolites was confirmed by investigations with [4,5,9,10-¹⁴C]pyrene.     

Monogènes Dactylogyridae parasites de Cyprinidae du genreBarbus d'Afrique du Nord

Resumé Quinze nouvelles espèces de Monogènes Dactylogyridae sont décrites chez quinze espèces deBarbus (Teleostei, Cyprinidae) appartenant aux sous-genresB. (Barbus) etB. (Labeobarbus) en Afrique du Nord. Les barbeaux examinés proviennent des différents bassins hydrographiques du Maroc et d'une localité nommée Hamman Bourgiba en Tunisie. Dans cette dernière région, le genreBarbus n'est représenté que par une seule espèce:Barbus (B.) callensis. Au Maroc, on en dénombre actuellement quatorze dont quatre appartiennent au sous-genreLabeobarbus: il s'agit deBarbus (L.) fritschii; B. (L.) harteti; B. (L.) paytonii etB. (L.) reinii. Les dix espèces appartenant au sous-genreBarbus sont:Barbus (B.) figuiensis; B. (B.) ksibi; B. (B.) lepineyi; B. (B.) magniatlantis; B. (B.) massaensis; B. (B.) moulouyensis; B. (B.) nasus; B. (B.) pallaryi; B. (B.) setivemensis etB. (B.) issenensis.Chaque sous-genre possède son propre pool parasitaire, à l'exception deDactylogyrus marocanus n. sp., recontré sur des espèces appartenant aux deux sous-genres (B. (L.) fritschii, B. (L.) paytonii, B. (L.) harteti, B. (L.) reinii, B. (L.) nasus, B. (B.) setivimensis, B. (B.) ksibi). Sur les cinqDactylogyrus parasitant lesLabeobarbus, trois présentent une spécificité stricte vis à vis de leur hôte. Il s'agit deDactylogyrus reinii n. sp. surB. (L.) reinii; D. volutus n. sp. etD. zatensis n. sp. surB. (L.) fritschii. Les espècesD. oumiensis n. sp. etD. kulindrii n. sp. présentent une spécifité stenoxène et parasitent respectivementB. (L.) harteti, B. (L.) paytonii, B. (L.) reinii etB. (L.) fritschii, B. (L.) reinii.Nous avons recontré neufDactylogyrus chez les poisson-hôtes appartenent au sous-genreBarbus. Six d'entre eux ont une spécificité oïoxène; ce sont:D. guirensis n. sp.,D. atlasensis n. sp. etD. draaensis n. sp. surB. (B.) pallaryi; D. borjensis n. sp. surB. (B.) nasus etD. heteromorphus n. sp. etD. tunisiensis n. sp. surB. (B.) callensis. Les trois autres parasites ont un spectre d'hôtes plus large. Il s'agit deD. ksibii n. sp. recontré chezB. (B.) ksibi, B. (B.) setivimensis etB. (B.) magniatlantis; D. ksibioïdes n. sp. recontré chezB. (B.) setivimensis etB. (B.) moulouyensis. L'espèceD. fimbriphallus n. sp. stenoxène, se recontre chez les poisson-hôtes du versant Sud de l'Atlas et de la façade méditerranéenne à savoir:B. (B.) figuiensis, B. (B.) lepineyi, B. (B.) massaensis, B. (B.) moulouyensis, B. (B.) pallaryi etB. (B.) issenensis.Le rôle des Dactylogyridae en tant que marqueurs biogéographiques, phylogénétiques et taxonomiques est discuté à partir de la composition spécifique des communautés de Monogènes rencontrés et de leurs différents types morphologiques.

---

Fifteen new species of the Dactylogyridae (Monogenea) parasitic on fifteen species of barbels (Barbus) from North Africa (Teleostei, Cyprinidae) are described. The fishes studied belong to two subgenera,B. (Labeobarbus) andB. (Barbus), collected from various hydrographical basins of Morocco and from the Hamman Bourgiba locality in Tunisia. In the latter area, the genusBarbus is represented by onlyBarbus (Barbus) callensis. In Morocco, fourteen species are listed, four of which belong to the subgenusLabeobarbus; these areBarbus (L.) fritschii; B. (L.) harteti; B. (L.) paytonii andB. (L.) reinii. The other ten species belong to the subgenusBarbus: these areBarbus (B.) figuiensis; B. (B.) ksibi; B. (B.) lepineyi; B. (B.) magniatlantis; B. (B.) massaensis; B. (B.) moulouyensis; B. (B.) nasus; B. (B.) pallaryi; B. (B.) setivimensis andB. (B.) issenensis. Each of the two subgenera has its unique parasitic fauna, except forDactylogyrus marocanus n. sp. collected on species belonging to both subgenera (B. (L.) fritschii, B. (L.) paytonii, B. (L.) harteti, B. (L.) reinii, B. (B.) nasus, B. (B.) setivimensis andB. (B.) ksibi). Of the five monogeneans found onLabeobarbus, three appear to be specific to one host: they areDactylogyrus reinii n. sp. onB. (L.) reinii, andD. volutus n. sp. andD. zatensis n. sp. onB. (L.) fritschii. D. kulindrii n. sp. parasitisedB. (L.) reinii andB. (L.) fritschii; andD. oumiensis n. sp. occurred onB. (L.) reinii, B. (L.) paytonii andB. (L.) harteti. NineDactylogyrus species were found in fishes belonging to the subgenusBarbus. Six of them have an oïoxenous specificity: these areD. guirensis n. sp.,D. atlasensis n. sp. andD. draaensis n. sp. onB. (B.) pallaryi; D. borjensis n. sp. onB. (B.) nasus andD. heteromorphus n. sp. andD. tunisiensis n. sp. on(B.) callensis. These other three have a wider range of hosts: they areD. ksibii n. sp. collected fromB. (B.) ksibi, B. (B.) setivimensis andB. (B.) magniatlantis, andD. ksibioïdes n. sp. found onB. (B.) setivimensis andB. (B.) moulouyensis. D. fimbriphallus n. sp. is a characteristic parasite of fishes from the southern side of the Atlas mountains and the Mediterranean coast (B. (B.) figuiensis, B. (B.) lepineyi, B. (B.) massaensis, B. (B.) moulouyensis, B. (B.) pallaryi andB. (B.) issenensis).The role of dactylogyrids as biogeographical phylogenetic and taxonomic indicators is discussed in relation to the specific structure of the monogenean communities and the different morphological types found.

     

Volume regulation in human fibroblasts: role of Ca2+ and 5-lipoxygenase products in the activation of the Cl− efflux

Trypsinized human skin fibroblasts in suspension perform regulatory volume decrease (RVD) after cell swelling in hypotonic medium. During RVD, ³⁶Cl^– efflux is dramatically increased and the cell membrane is depolarized, indicating the activation of Cl^– channels. This activation of Cl^– channels depends on extracellular as well as on intracellular Ca²⁺. The swelling-induced Cl^– efflux and the RVD response are inhibited by the 5-lipoxygenase inhibitor ETH 615-139. Finally, following hypotonic treatment, cellular pH decreases. The pH decrease does not involve the Cl^–/HCO ₃ ^– exchange because it is independent of the external Cl^– concentration.T. Mastrocola was recipient of a scientific fellowship from the Italian Consiglio Nazionale delle Ricerche (C.N.R.). This work was supported by Progetto Finalizzato Ingegneria Genetica, C.N.R., Roma, and by the Danish Natural Research Council.     

The 23-kilodalton E1 phosphoprotein of bovine papillomavirus type 1 is nonessential for stable plasmid replication in murine C127 cells.

The 23-kDa protein encoded by the 5' segment of the E1 open reading frame of bovine papillomavirus type 1 (BPV1) was previously ascribed a negative regulatory function for the replication of viral plasmid DNA. However, results from recent functional and biochemical studies do not readily support this genetic assignment. Therefore, we have reassessed the role of this protein in papillomavirus DNA replication by using a mutant of BPV1 which is unable to express this E1 protein. This mutant viral DNA was found to replicate extrachromosomally with stability and copy number per cell similar to those of wild-type plasmid DNA. Thus, the absence of expression of the 23-kDa E1 protein did not lead to deregulated viral plasmid replication. We conclude that the 23-kDa E1 protein is nonessential for stable plasmid replication.