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Fas and Fas-associated death domain (FADD) play a critical role in the homeostasis of different cell types. The regulation of Fas and FADD-mediated cell death is pivotal to many physiological functions. The activation of T lymphocytes by concanavalin A (Con A) inhibited Fas-mediated cell death. We identified that among the several activation signals downstream of Con A stimulation, mitogen-activated protein (MAP) kinase kinase (MKK) was the major kinase pathway that antagonized Fas-triggered cell death. MKK1 suppressed FADD- but not caspase-3– induced apoptosis, indicating that antagonism occurred early along the Fas-initiated apoptotic cascade. We further demonstrated that activation of MKK1 led to expression of FLIP, a specific inhibitor of FADD. MKK1 inhibition of FADD-induced cell death was abrogated if induction of FLIP was prevented, indicating that FLIP mediates MKK1 suppression of FADD-mediated apoptosis. Our results illustrate a general mechanism by which activation of MAP kinase attenuates apoptotic signals initiated by death receptors in normal and transformed cells.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core‐related antigens (HBc/HBeAg) if well‐differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH‐7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH‐7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus‐associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS‐‐PAGE after labeling with [35S]methionine. Antisera specific for X‐gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV‐related gene function and HBV replication.

Penicillium and Aspergillus species have been identified as prevalent indoor airborne fungi that are associated with extrinsic bronchial asthma. We have recently analyzed the IgE–binding components in 8 prevalent Penicillium and Aspergillus species (P. citrinum, P. notatum, P. oxalicum, P. brevicompactum, A. fumigatus, A. flavus, A. oryzae and A. niger) by immunoblotting and N–terminal amino acid sequence analysis. Our results show that the alkaline and/or vacuolar serine proteinases are the major allergens in these prevalent fungal species. IgE cross–reactivity among these major allergens was also detected. Results obtained provide an important basis for clinical allergy. In addition, monoclonal antibodies against alkaline and/or vacuolar serine proteinase allergens have been generated. These antibodies can be applied for the standardization of allergenic extracts.

A new hepatitis B virus (HBV) transcript of about 2.2 kilobases was identified in HBV DNA-transfected human hepatoma cells. The 5' terminus of this viral RNA appears to map at one or more of the precore initiation sites, contains a deletion of 1,223 bases corresponding to the last codon of the core gene to the middle of the surface antigen gene, and terminates at the 3' polyadenylation site used by the other known HBV RNAs. The junction region of the deleted sequences showed the conserved splice donor and acceptor GT-AG sequences. Moreover, when a mutant HBV DNA in which the splice acceptor site was changed from AG to CG was transfected into human hepatoma cells, no 2.2-kilobase RNA was detected, further suggesting that this RNA represents a spliced transcript. The core gene, although an amino acid shorter, still encoded a functional viral core protein in complementation experiments. Sequence analysis of the cDNA of the 2.2-kilobase RNA suggests that this transcript can potentially encode a new protein that comprises the reverse transcriptase domain of HBV. However, genetic analysis using a transient DNA transfection system suggests that the gene product(s) of this transcript is not essential for viral replication. The function of this transcript remains to be studied.

Results obtained in the present study suggest that the 33 kD major allergen of P. citrinum may be an alkaline serine proteinase.

Background:Penicillium chrysogenum (Penicillium notatum) is a prevalent airborne Penicillium species. A 34-kD major IgE-reacting component from P. chrysogenum has been identified as an alkaline serine protease (Pen ch 13, also known as Pen n 13 before) by immunoblot and N-terminal amino acid sequence analysis. Methods: In the present study, Pen ch 13 was further characterized in terms of cDNA cloning, protein purification, enzymatic activity, histamine release and IgE cross-reactivity with alkaline serine protease allergens from two other prevalent fungal species – P. citrinum (Pen c 13) and Aspergillus flavus (Asp fl 13). Results: A 1,478-bp cDNA (Pen ch 13) that encodes a 398-amino-acid alkaline serine protease from P. chrysogenum was isolated. This fungal protease has pre- and pro-enzyme sequences. The previously determined N-terminal amino acid sequence of the P. chrysogenum 34-kD major allergen is identical to that of residues 116–125 of the cDNA. Starting from Ala116, the deduced amino acid sequence (283 residues) of the mature alkaline serine protease has a calculated molecular mass of 28.105 kD with two cysteines and two putative N-glycosylation sites. It has 83 and 49% sequence identity with the alkaline serine proteases from P. citrinum and A. fumigatus, respectively. The recombinant Pen ch 13 was recovered from inclusion bodies and isolated under denaturing condition. This recombinant protein reacted with IgE antibodies in serum from an asthmatic patient and with monoclonal antibodies (PCM8, PCM10, PCM39) that reacted with the 34-kD component from P. chrysogenum. The N-terminal amino acid sequence of the purified native Pen ch 13 is identical to that determined previously for the 34-kD major allergen in crude P. chrysogenum extracts. The purified native Pen ch 13 has proteolytic activity with casein as the substrate at pH 8.0. This enzymatic activity was inhibited by phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Pen ch 13 was also able to degrade gelatin and collagen but not elastin. Basophils from 5 asthmatic patients released histamine (12–73%) when exposed to the purified Pen ch 13. In ELISA (enzyme-linked immunosorbent assay) experiments, IgE for Pen ch 13 was able to compete with purified Pen ch 13, Pen c 13 or Asp fl 13 in a dose-related manner. Conclusions: These results demonstrated that the 34-kD major allergen of P. chrysogenum is an alkaline serine protease. These results also indicated that atopic patients primarily sensitized by either of these prevalent fungal species may develop allergic symptoms by exposure to other environmental fungi due to cross-reacting IgE antibodies against this protease.

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