Aberrant caspase-activated DNase (CAD) transcripts in human hepatoma cells.

The gene of caspase-activated DNase (CAD), the key enzyme for nucleosome cleavage during apoptosis, is mapped at chromosome 1p36, a region usually associated with hemizygous deletions in human cancers, particularly in hepatoma (HCC). It is tempting to speculate that CAD plays a tumour-suppressive role in hepatocarcinogenesis. To address this, we examined the CAD transcripts in six human HCC cell lines, one liver tissue from a non-HCC subject, and peripheral blood leukocytes (PBL) from three healthy individuals. Alternatively spliced CAD transcripts with fusion of exon 1 to exon 7 were isolated in most of the examined samples including HCC cells and normal controls. However, relatively abundant alternatively spliced CAD transcripts with fusion of exon 2 to exon 6 or 7, in which the corresponding domain directing CAD interaction with ICAD was preserved, were found only in poorly differentiated Mahlavu and SK-Hep1 cells. Interestingly, an abnormal CAD transcript with its exon 3 replaced by a truncated transposable Alu repeat was isolated in Hep3B cells, indicative of the implication of an Alu-mediated genomic mutation. Moreover, mis-sense mutations in the CAD genes were identified in all six HCC cell lines. Upon UV-induced apoptosis, DNA fragmentation efficiency was found to be intact, partially reduced and remarkably reduced in Huh7 and J328, Hep3B and HepG2, and Mahlavu cells, respectively. That mutations and aberrantly spliced transcripts for the CAD gene are frequently present in human HCC cells, especially in poorly differentiated HCC cells, suggests a significant role of CAD in human hepatocarcinogenesis.

Pulmonary function changes and immunomodulation of cytokine expression by zafirlukast after sensitization and allergen challenge in brown Norway rats.

BACKGROUND: The cysteinyl leukotrienes are known important mediators in bronchial asthma. OBJECTIVE: Our purpose was to evaluate the effect of zafirlukast on the late-phase reaction, bronchial hyper-responsiveness (BHR) and T cell-related cytokine mRNA expression in ovalbumin (OA)-sensitized brown Norway rats (BNRs). METHODS: Thirty BNRs were equally divided into three groups. Group I and II animals were sensitized and then provoked with OA. Zafirlukast was given intraperitoneally (i.p.) to group I animals prior to provocation. Group II animals received i.p. normal saline. Group III animals (controls) were not sensitized and breathed aerosolized saline. After OA provocation, the animals were anaesthetized. Pulmonary function tests (PFT) were performed at baseline and after varying doses of acetylcholine. Thereafter, bronchoalveolar lavage (BAL) was performed and the lungs were examined histologically. Total RNA was extracted from lung tissue and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using primers for IL-2, IL-4, IL-5, IL-6, IL-10, TNF-alpha, IFN-gamma, iNOS and beta-actin. RESULTS: Group II OA-treated BNRs had worse PFT results, more severe bronchoconstriction in response to acetylcholine, and more severe inflammation in lung tissue than the other two groups. Group II had higher IL-2, IL-4, IL-10 and IFN-gamma cytokine levels in BAL fluid and higher IL-2, IL-4, IL-5, IL-6, IL-10, IFN-gamma, TNF-alpha and iNOS mRNA levels when compared with group I. CONCLUSION: Zafirlukast is effective in preventing late-phase bronchoconstriction and BHR, reducing inflammatory response, and decreasing IL-2, IL-4, IL-5, IL-6, IL-10 and IFN-gamma and iNOS mRNA expression.

Molecular cloning and expression of a Penicillium citrinum allergen with sequence homology and antigenic crossreactivity to a hsp 70 human heat shock protein.

BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. However, detailed studies on allergens of this ubiquitous Penicillium species are still lacking. OBJECTIVE: For the characterization of allergens of this prevalent Penicillium species, molecular cloning and expression of the allergen genes of P. citrinum were performed in the present study. METHODS: Molecular cloning of the allergen genes was performed by using a lambda Uni-Zap XR cDNA library of P. citrinum and serum from an asthmatic patient. The cloned cDNA was excised from the phage vector as a recombinant pBluescript phagemid and sequenced. The cDNA of the IgE-binding clone was expressed as fusion protein with the glutathione-S-transferase. The corresponding natural allergen was analysed by absorption immunoblotting using monoclonal antibody and serum from asthmatic patient. The frequency of IgE-binding to the allergen cloned was analysed by dot immunoassay using recombinant allergen and by immunoblotting using the whole extract of P. citrinum. RESULTS: In the screening of cDNA library of P. citrinum using serum from an asthmatic patient, IgE-binding cDNA clones designated SC4 and XL were obtained. The 5'-truncated, 0.7-kb and 1.7-kb cDNA inserts of clones SC4 and XL contained open reading frames of 163 and 503 amino acids, respectively. On alignment, the deduced amino acid sequences showed that 97 (60%) of the 163 amino acids and 376 (75%) of the 503 amino acids were identical to the corresponding amino acid sequence of the human heat shock protein in the hsp70 family. Both recombinant SC4 and XL showed positive SDS-PAGE-immunoblot reactivity to a monoclonal antibody MA3-006 against the human hsp 70 protein. For characterization of the corresponding natural allergen, immunoblotting reactivities of MA3-006 and IgE antibodies to the 70 kDa component of P. citrinum have been shown to be disappeared after absorption of these antibodies with the recombinant SC4 protein. Sera from 14 (41%) of 34 Penicillium-allergic patients showed IgE-binding to the recombinant XL protein and the 70 kDa component in the extract of P. citrinum. CONCLUSION: Results obtained suggest that hsp 70 is an allergen of P. citrinum and that clones SC4 and XL contain partial cDNAs of this allergen gene.

Characterization of the 33-kilodalton major allergen of Penicillium citrinum by using MoAbs and N-terminal amino acid sequencing.

BACKGROUND: The 33 kD component has been identified as a major allergen of Penicillium citrinum, the most prevalent Penicillium species in the Taipei area of Taiwan. OBJECTIVE: This study analyses the isoforms, antigenic cross-reactivity and the N-terminal amino acid sequence of the 33 kD allergen of P. citrinum. METHODS: The composition of isoforms and antigenic cross-reactivity was analysed by SDS-PAGE and 2D-immunoblotting using MoAbs generated. The N-terminal sequence was analysed by using an automatic gas/liquid phase sequencer. RESULTS: Two MoAbs (55A and 34H) against the 33 kD allergen were generated in the present study. In addition to the 33 kD component, MoAb 34H also showed immunoblot reactivity to other components in the crude extract of P. citrinum. Analysed by 2D-immunoblotting, at least six different isoforms of the 33 kD component with pI values ranging from 6.75 to greater than 7.0 were shown to be reactive to both MoAbs and IgE antibodies in serum of an asthmatic patient. Different immunoblot patterns were observed when both MoAbs were reacted with four different strains of P. citrinum used in the present study. Among another six different Penicillium and four different Aspergillus species tested, only an immunoblot reactivity of MoAb 55A to the 33 kD component of P. brevicompactum was observed. In 2D-immunoblotting, components of P. brevicompactum with an MW of about 33 kD and pI values similar to those of the 33 kD component of P. citrinum reacted with MoAb 55A and IgE antibodies in serum of the asthmatic patient. The N-terminal amino acid sequence of the 33 kD component of P. citrinum was determined to be ANVVQSNVP which was identical to the first 9 N-terminal amino acids of a heat-labile alkaline serine proteinase from P. citrinum. CONCLUSION: Results obtained in the present study suggest that the 33 kD major allergen of P. citrinum may be an alkaline serine proteinase.

Allergenic components in three different species of Penicillium: crossreactivity among major allergens.

BACKGROUND: Penicillium species have been considered as important causative agents of extrinsic bronchial asthma. However, little is known about the allergens of these ubiquitous airborne fungal species. OBJECTIVE: This study compares the allergenic profiles and allergenic crossreactivity among allergens of three prevalent airborne Penicillium species. METHODS: IgE-binding Penicillium components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)-immunoblotting using sera from 67 asthmatic patients. The presence of allergenic crossreactivity was analysed by immunoblot inhibition. RESULTS: Among the 67 serum samples tested, 15, 14 and 11 samples showed IgE reactivity to components of P. citrinum, P. notatum and P. brevicompactum, respectively. All 15 P. citrinum-positive serum samples showed IgE-binding to a 33 kDa extract component of this species. Thirteen (93%) of the 14 P. notatum-positive serum samples and 10 (91%) of the 11 P. brevicompactum-positive sera also showed IgE reactivity to components with a molecular weight of about 33 kDa in individual Penicillium species. All of the 10 P. brevicompactum 33 kDa component-positive serum samples showed IgE reactivity to the 33 kDa components of the other two Penicillium species tested. Dose-dependent inhibition of IgE-binding to these major allergens was observed when the positive serum sample was absorbed with different amounts of individual allergenic extract as well as with different amounts of extracts prepared from the other two Penicillium species. CONCLUSION: Although different allergenic profiles were observed in the three different Penicillium species tested, results showed that there was an IgE crossreactivity among the 33 kDa group major allergens of P. citrinum, P. notatum and P. brevicompactum.

Molecular cloning of cDNA coding for the 68 kDa allergen of Penicillium notatum using MoAbs.

To characterize the 68 kDa allergen of Penicillium notatum (also known as P. chrysogenum), a molecular antibody (MoAb) (P40) was previously generated. For cDNA cloning, three more MoAbs (3F, 5A3, 5G2) were generated in the present study. A mixture of all the four MoAbs was used in cloning of the gene coding for the 68 kDa allergen from a lambda gt11 cDNA library of P. chrysogenum. A cDNA clone (A6) with DNA insert of about 0.5 kb which encodes for the 3'-terminal nucleotide sequence of the 68 kDa allergen was obtained. The cloned sequence contained two putative N-glycosylation sites. The reduction in molecular weight from 68 to 62 kDa in immunoblotting after treatment of the crude extract of P. notatum with N-glycosidase F indicates that the 68 kDa allergen is a glycoprotein. Nucleotide sequence determination showed that 188 (54%) of the 348 nucleotides of the cDNA sequence obtained were identical to the same region of the nucleotide sequence of the beta-N-acetylglucosaminidase gene of Candida albicans. Although the cDNA clone obtained did not encode the full-length gene of the 68 kDa allergen, polypeptide expressed from the A6 cDNA showed positive immunological reactivities to all four MoAbs used in the cloning experiment and to IgE antibodies in sera of asthmatic patients. There was a loss of immunoblotting activity to the 68 kDa component after absorption of MoAb P40-containing culture supernatant with filters blotted on plaque lawns of cDNA clone A6. Moreover, the immunoblotting activity remained when the MoAbs affinity-purified with filters containing polypeptides encoded by the cDNA insert of clone A6 were used.(ABSTRACT TRUNCATED AT 250 WORDS)