Patients' Reports of Traumatic Experience and Posttraumatic Stress in Psychiatric Settings.

OBJECTIVE: To determine the prevalence of traumatic experience (TE) among patients in psychiatric settings in Hong Kong and the associations between TE and levels of distress and anxiety and depressive symptoms. METHODS: 129 patients who have received inpatient psychiatric services were recruited. Their lifetime TE was assessed using the Life Event Checklist (LEC), and TE in psychiatric settings using the Psychiatric Experiences Questionnaire (PEQ). Their level of distress symptoms was assessed using the Impact of Event Scale-Revised (IES-R), and the level of anxiety and depressive symptoms using the Hospital Anxiety and Depression Scale (HADS). RESULTS: The prevalence of direct and indirect TE was 84.5%, as was the prevalence of TE in psychiatric settings. Common TE in psychiatric settings included witnessing another patient being taken down (61.2%), being put in restraints of any kind (41.1%), and witnessing another patient being physically assaulted by another patient (36.4%). TE in psychiatric settings associated with high prevalence of severe or extreme distress 1 week after the event included being forced to take medication against their will (52.2%), being threatened with physical violence (52.2%), and experiencing a physical assault (50.0%). Lifetime TE (the total number of LEC items reported) was associated with severity of distress and anxiety and depressive symptoms, whereas TE in psychiatric settings (the total number of PEQ items reported) was associated with severity of distress only. The total number of LEC items reported is the only predictor of levels of distress and anxiety and depressive symptoms. CONCLUSIONS: Lifetime TE and TE in psychiatric settings are common among patients with SMI. Trauma-informed care is suggested for mental health services.

Validation of the Hong Kong Cantonese Version of World Health Organization Five Well-Being Index for People with Severe Mental Illness.

OBJECTIVE: The World Health Organization Five Well-Being Index (WHO-5) has been developed to measure psychological wellbeing. Translation and linguistic validation of the WHO-5 into a Cantonese version has been accomplished for local use but it is not yet validated in people with severe mental illness in Hong Kong. This study aimed to examine the applicability of WHO-5 in measuring the psychological wellbeing dimension of people with severe mental illness. A brief and easily administrated tool to measure psychological wellbeing of people with severe mental illness can be used to provide an outcome measure in research studies and clinical trials. METHODS: Subjects were randomly recruited from the Extended-Care Patient Intensive Treatment, Early Diversion and Rehabilitation Stepping-Stone Project (EXITERS) and the Rehabilitation Activity Centre (RAC) of Kwai Chung Hospital in Hong Kong. They were invited to complete the abbreviated version of Hong Kong Chinese World Health Organization Quality of Life (WHOQOL-BREF [HK]) and WHO-5 (Cantonese version) separately and concurrent validity was examined. RESULTS: A total of 84 subjects were recruited, 42 each from EXITERS and RAC. In all, 49 (58%) were male and 35 (42%) were female. The mean ± standard deviation age was 43.2 ± 9.7 years. Their mean duration of mental illness was 16.4 ± 10.5 years and the mean years of education was 10.17 ± 2.5 years, i.e. about junior secondary school level in Hong Kong. The internal consistency of the WHO-5 was satisfactory (0.86) and was comparable with previous reports. Regarding validity, 1-factor structure with an eigenvalue of 3.24 explained 64.8% of total variance of WHO-5 for people with severe mental illness. Concurrent validity was established with moderate correlation (0.41-0.51) between WHO-5 and 4 domains of the WHOQOL-BREF (HK). CONCLUSION: The WHO-5 (Cantonese version) is a reliable and valid tool to assess the psychological wellbeing of people with severe mental illness in Hong Kong. It can be used to monitor the effectiveness of psychological intervention aimed at improving the wellbeing of such patients.

Single-molecule imaging of BMP4 dimerization on human periodontal ligament cells.

We expressed bone morphogenetic protein 4 (BMP4) fused with enhanced green fluorescent protein (BMP4-EGFP) in the secretory pathways of producer cells. Fluorescent EGFP was acquired only after we interrupted the transport of BMP4-EGFP by culturing cells at a lower temperature (20°C), and the dynamics of BMP4-EGFP could be monitored by single-molecule microscopy. Western blotting analysis confirmed that exposure to low temperature helped the integrated formation of BMP4-EGFP fusion proteins. In this study, for the first time, we could image the fluorescently labeled BMP4 molecules localized on the plasma membrane of living hPDL cells. The one-step photobleaching with EGFP and the "blinking" behavior of quantum dots suggest that the fluorescent spots represent the events of single BMP4 molecules. Single-molecule tracking showed that the BMP receptors (BMPR) dimerize after BMP4 stimulation, or that a complex of one BMP4 molecule and a pre-formed BMPR dimer develops first, followed by the binding of the second BMP4 molecule. Furthermore, BMP4-EGFP enhanced the osteogenic differentiation of hPDL cells via signal transduction involving BMP receptors. This single-molecule imaging technique might be a valuable tool for the future development of BMP4 gene therapy and regenerative medicine mediated by hPDLs.

Colour photographs for screening in neovascular age-related macular degeneration: are they necessary?

AIMS: To investigate whether optical coherence tomography (OCT) with associated infra-red images provide enough information to determine treatment decisions in the management of neovascular age-related macular degeneration (nAMD), or whether retinal colour photography is also necessary. METHODS: In all, 87 OCT scans of 82 eyes with nAMD undergoing monitoring post ranibizumab treatment were taken using the Zeiss Stratus (Carl Zeiss Meditec, Jena, Germany; n=87) together with their corresponding infra-red images. Fundus colour photographs were also taken. These images were reviewed by an experienced assessor, and a ranibizumab treatment decision was made during a multidisciplinary team retinal image review meeting. RESULTS: In all, 30 OCT scans (34.5%) showed intraretinal or subretinal oedema. A total of 24 colour photographs (19.5%) demonstrated retinal haemorrhage. Corresponding OCT infra-red images gave poor sensitivity in detecting haemorrhages (0.176). In 16.7% of decisions to treat, haemorrhage alone was the deciding factor. Signs of disease activity seen only on colour photography were the deciding factor in clinical decisions for 8% of scans assessed. CONCLUSIONS: The presence or increase of intra-retinal oedema is an important sign of activity triggering ranibizumab retreatment, but some eyes show signs of retinal haemorrhage without coexisting oedema. These haemorrhages are often only seen on either colour imaging or fundoscopy and are unclear or invisible on OCT scans and infra-red images. Therefore, although retinal colour photography creates additional expense, it is indispensable for making informed retreatment decisions, if patients are monitored using retinal imaging alone.

Highly efficient multipotent differentiation of human periodontal ligament fibroblasts induced by combined BMP4 and hTERT gene transfer.

Because periodontal ligament (PDL) cells are reported to contain progenitor or stem cell populations, they are considered a beneficial cell source for clinical periodontal regeneration. Both bone morphogenetic protein 4 (BMP4) and human telomerase reverse transcriptase (hTERT) have essential roles in the modulation of stem cell properties. In this study we report for the first time that the combined ectopic expression of BMP4 and hTERT significantly enhanced the multipotent differentiation efficiency and capacity of human PDL fibroblasts (PFs), as shown by osteogenic, adipogenic and neurogenic differentiation in vitro, and cementum/PDL-like tissue regeneration in vivo. These findings may be attributed, at least in part, to the original upregulation of important stem cell markers, such as scleraxis, Stro-1 and CD146, and the extremely lowered threshold for BMP concentration to activate BMP signaling by enhanced basal phosphorylation levels of Smad 1/5/8. In addition, the significantly reduced expression levels of CD146 and CD90 with the presence of Noggin confirms the direct effect of BMP4 on the stem cell-like phenotype of genetically modified PF cells (BT-PFs). Furthermore, BT-PFs exhibited a high neural differentiation capacity (>75%). After transplantation into NOD/SCID mice, genetically modified-PFs generated cementum/PDL-like structures on the surface of the carrier. The multipotency of these modified cells potentially provides an attractive source of stem cells for therapeutic purposes and regenerative medicine.

Analysis of protein profiles in human epithelial ovarian cancer tissues by proteomic technology.

BACKGROUND: Screening in ovarian cancer is progressively finding out candidate genes and proteins which may work as screening biomarkers and play a role in tumor progression. We examined the protein expression patterns of ovarian cancer tissues using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of fight mass spectrometry (MALDI-TOF MS). METHODS: Tissues from 36 ovarian cancers and 20 normal ovaries were examined by 2-DE. The images of silver stained gels were analyzed by ImageMaster 2D Elite. The peptide mixtures, after in-gel digestion, were determined by MALDI-TOF MS for fingerprinting. The de-isotope tryptic peptide profiles were matched by using the Mascot search engine based on the entire NCBI and Swiss-Prot protein databases. Western/dot blots were then applied to verify the findings. RESULTS: In ovarian cancer, 12 proteins that showed differential expressions were identified unequivocally. Among these proteins, five proteins (galectin-1, cathepsin B, ubiquitin carboxy-terminal hydrolase L1, HLA class II antigen DRB1-11 and heat shock protein 27) were up-regulated and seven proteins (cellular retinol-binding protein, transthyretin, SH3 binding glutamic-rich-like protein, tubulin-specific chaperone A, DJ-1, gamma-actin and tropomyosin 4) were down-regulated. CONCLUSION: The present study is the first to report the up-regulation of ubiquitin carboxy-terminal hydrolase L1 and the down-regulation of SH3 binding glutamic-rich-like protein, tubulin-specific chaperone A, and tropomyosin 4 in human ovarian cancer tissues. Further cloning and functional analysis of these salient proteins will provide more information on their pathophysiologic roles in ovarian cancer.

Proteomic identification of membrane proteins regulating antimicrobial peptide resistance in Vibrio parahaemolyticus.

AIMS: To identify proteins regulating antimicrobial peptide (AMP) resistance in Vibrio parahaemolyticus using membrane subproteome analysis. METHODS AND RESULTS: Three synthetic AMPs (Q4, Q6 and H1) and a natural one from fish (pleurocidin) were used for selection of AMP-resistant strains. Differential expression patterns of the outer and inner membrane proteins (OMPs and IMPs) among wild-type and the resistant strains were obtained using two-dimensional gel electrophoresis. Two OMPs (TolC and flagellin) and five IMPs [transcription termination factor (NusA), long-chain fatty acid transport protein (FadL), elongation factor Tu (EF-Tu), ATP synthase F1, alpha subunit (F1-ATPa) and dihydrolipoamide dehydrogenase (DLD)] were identified using LC-ESI-Q-TOF MS/MS and Mascot program. Real-time quantitative polymerase chain reaction was also performed to determine the mRNA expression level of the target genes. All seven membrane proteins except FadL were upregulated in the AMP-resistant clones, both in the translational and transcriptional levels. CONCLUSIONS: Our results suggested that V. parahaemolyticus may obtain their resistance against AMPs through upregulation of the multidrug efflux transporter, effective repair of damaged membranes and prevention of cellular penetration of AMPs. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing bacterial AMP resistance mechanism using proteomic methodologies. Elucidating the mechanism could help in the development of more sustainable antimicrobial agents.

Salt-responsive outer membrane proteins of Vibrio anguillarum serotype O1 as revealed by comparative proteome analysis.

AIMS: Vibrio anguillarum is a universal marine pathogen causing vibriosis. Vibrio anguillarum encounters different osmolarity conditions between seawater and hosts, and its outer membrane proteins (OMPs) play a crucial role in the adaptation to changes of the surroundings. In the present study, proteomic approaches were applied to investigate the salt-responsive OMPs of V. anguillarum. METHODS AND RESULTS: Lower salinity (0.85% NaCl) is more suitable for growth, survival and swimming motility of the bacterium. Comparative two-dimensional electrophoresis (2-DE) analysis reveals six differentially expressed protein spots among three different salinities, which were successfully identified as OmpU, maltoporin, flagellin B, Omp26La, Omp26La and OmpW respectively. CONCLUSIONS: OmpW and OmpU were highly expressed at 3.5% salinity, suggesting their role in the efficient efflux of NaCl. Maltoporin was downregulated in higher salinity, indicating that higher osmolarity inhibits carbohydrate transport and bacterial growth. Omp26La, the homologue of OmpV, functions as a salt-responsive protein in lower salinity. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report describing salt stress-responsive proteins of V. anguillarum using proteomic approaches. Our results provide a useful strategy for delineating the osmoregulatory mechanism of the marine pathogens.

Kinetics and functional assay of liver repopulation after human cord blood transplantation.

BACKGROUND AND AIMS: To evaluate donor cell engraftment and the kinetics of cell repopulation in the injured mouse liver following human umbilical cord blood cell transplantation. METHODS: Nonobese diabetic/severe immunodeficient mice were treated with allyl alcohol to induce liver injury. Twenty-four hours later, umbilical cord blood derived mononuclear cells were transplanted by intra-splenic injection. Mice were sacrificed from 1 to 180 days after transplantation. Temporal changes in the ratio of human cells and fluorescence counts of human sex-determining region Y alleles in mouse liver were determined to evaluate the kinetics of cell repopulation. Mouse liver and sera were examined for the presence of human albumin. RESULTS: Human cell repopulation was extremely rapid in the first week following transplantation, with a doubling time of 1.16-1.39 days apparent. Thereafter cell doubling rate slowed significantly. Cells displaying characteristics of human hepatocytes were still evident at 180 days. Human albumin was detected in mouse liver and sera. CONCLUSION: These findings confirm those from previous studies demonstrating that cells derived from human umbilical cord blood have the capacity to differentiate into cells with human hepatocyte characteristics in mouse liver following injury. Moreover, the detailed information collected regarding the kinetics of human cell repopulation in mouse liver will be of relevance to future studies examining the use of umbilical cord blood cells in liver transplantation therapy.

Orbitofrontal cholesterol granuloma.

Cholesterol granuloma of the orbital bones is a rare but readily recognisable condition. It is an osteolytic lesion with a granulomatous reaction surrounding cholesterol crystals, old haemorrhage and a fibrous capsule. There is a male preponderance and it usually occurs in young or middle-aged men. It is treatable with drainage and curettage via an orbitotomy, and craniotomy or wide bone removal is almost never required. Six cases of this condition were reviewed to highlight the typical clinical presentation, computed tomography and magnetic resonance results, and surgical management.

Scedosporium endophthalmitis: two fatal disseminated cases of Scedosporium infection presenting with endophthalmitis.

The incidence of disseminated infection with Scedosporium species is increasing in patients with haematological malignancy. Two fatal cases are reported of patients with acute myeloid leukaemia and neutropenia who presented with Scedosporium endophthalmitis. Diagnosis of fungal infection was delayed as blood and vitreous cultures were positive only after 3 days in patient 1 and blood culture was positive at 7 days in patient 2. Despite antifungal therapy with amphotericin B and additional fluconazole in patient 2, both patients died of overwhelming fungal septicaemia. Post-mortem examination of the right globe in patient 1 showed haemorrhagic necrotizing chorioretinitis with numerous fungal hyphae in choroidal vessels, choroid, retina and vitreous. Scedosporium species are often resistant to conventional antifungal therapy including amphotericin B. Diagnosis is difficult and mortality in disseminated infection is high.

Carbohydrate specificity of a toxic lectin, abrin A, from the seeds of Abrus precatorius (jequirity bean).

To elucidate of the mechanism of intoxication, the affinity of a toxic lectin, abrin A, from the seeds of Abrus precatorius for mammalian carbohydrate ligands, was studied by enzyme linked lectinosorbent assay and by inhibition of abrin A-glycan interaction. From the results, it is concluded that: (1) abrin A reacted well with Gal beta1-->4GlcNAc (II), Gal alpha1-->4Gal (E), and Gal beta1-->3GalNAc (T) containing glycoproteins. But it reacted weakly with sialylated gps and human blood group A,B,H active glycoproteins (gps); (2) the combining site of abrin A lectin should be of a shallow groove type as this lectin is able to recognize from monosaccharides with specific configuration at C-3, C-4, and deoxy C-6 of the (D)Fuc pyranose ring to penta-saccharides and probably internal Gal alpha,beta-->; and (3) its binding affinity toward mammalian structural features can be ranked in decreasing order as follows: cluster forms of II, T, B/E (Gal alpha1-->3/4Gal) > monomeric T > monomeric II > monomeric B/E, Gal > GalNAc > monomeric I >> Man and Glc (inactive). These active glycotopes can be used to explain the possible structural requirements for abrin A toxin attachment.

Structural characterization of the 60-kDa bermuda grass pollen isoallergens, a covalent flavoprotein.

Our studies suggest a tripartite structure for the 60-kDa allergen of Bermuda grass pollen (BG60) including a short N-terminal segment, a FAD-binding domain, and a C-terminal domain. The lower molecular weight isoallergens lack the N-terminal segment. The higher protease susceptibility and the lower melting temperature of approximately 20 degrees C of the lower molecular weight isoforms suggest that the N-terminal segment is essential for a compact structure. Database screening reveals that the protease-digested peptide sequences (approximately 180 residues in total) share 40% identity with the plant berberine bridge enzymes. In particular, a 24-residue peptide sequence displays high similarity to a conserved FAD-binding motif. The spectroscopic and SDS-PAGE analyses suggest that the cofactor FAD is covalently linked to the central domain. Therefore, we conclude that BG60 is identified as the first flavinylated allergen.

Arsanilic acid-Sepharose chromatography of pyruvate kinase from KB cells.

In the present study, arsanical-based affinity chromatography for pyruvate kinase (PK) isolation was explored. p-Arsanilic acid (4-aminophenyl arsonic acid), which contains an arsonic acid moiety structurally similar to inorganic pentavalent arsenate, was conjugated to Sepharose 4B via its para-amino group to form an As(V)-Sepharose matrix. The cellular proteins from KB cells bound to arsonic acid moieties were eluted by 50 mM sodium arsenate in Tris-HCl buffer (50 mM, pH 7.6). A single protein band with a molecular mass of 58 kDa was shown on a sodium dodecyl sulfate-polyacrylamide gel. By immunoblotting, amino acid sequencing and enzymatic analysis, the sodium arsenate-eluted 58-kDa protein was demonstrated to be a human PK (type M2). By using this one-step As(V)-Sepharose chromatography, PK from KB cells was purified 35.4-fold with a specific activity of 153.15 U/mg protein in the presence of 6 mM fructose-1,6-biphosphate. Although PK was eluted from an As(V)-Sepharose column with sodium arsenate, PK activity was apparently inhibited by the used eluent system, but not by p-arsanilic acid, indicating a specific interaction of As(V) to PK. In summary, our results indicate that As(V)-Sepharose can serve as a simple and efficient chromatographic support for PK purification from KB cells.

Identification and expression of an allergen Asp f 13 from Aspergillus fumigatus and epitope mapping using human IgE antibodies and rabbit polyclonal antibodies.

The Aspergillus genus of fungi is known to be one of the most prevalent aeroallergens. On two-dimensional immunoblotting using patients' sera containing IgE specific for Asp f 13, an allergen with a molecular mass of 33 kDa and a pI of 6.2 was identified. This allergen was also present in A. fumigatus culture filtrates. Furthermore, the sequence of the Asp f 13 cDNA was identical to that for alkaline protease isolated from A. fumigatus and showed 42-49% identity of amino acids with two proteases from P. cyclopium and T. album and with the Pen c 1 allergen from P. citrinum. Asp f 13 coding sequences were expressed in Escherichia coli as a [His](6)-tagged fusion protein which was purified by Ni(2+)-chelate affinity chromatography. Recombinant Asp f 13 was recognized by rabbit polyclonal antibodies against Asp f 13 and by IgE antibodies from subject allergic to A. fumigatus. To identify and characterize the linear epitopes of this allergen, a combination of chemical and enzymatic cleavage and immunoblotting techniques, with subsequent N-terminal sequencing and mass spectrometry, were performed. At least 13 different linear epitopes reacting with the rabbit anti-Asp f 13 antiserum were identified, located throughout the entire molecule. In contrast, IgE from A. fumigatus-sensitive patients bound to three immunodominant epitopes at the C-terminal of the protein.

Characterization of Pen n 13, a major allergen from the mold Penicillium notatum.

Penicillium notatum is a well-known indoor aeroallergen and is frequently included in skin test panels for allergic diagnosis. On two-dimensional immunoblotting using patients' sera containing IgE and monoclonal antibody D7B8 specific for Pen c 1 of P. citrinum, two allergens with a molecular mass of 33 kDa but different isoelectric points were identified. A novel cDNA coding for Pen n 13 was cloned and sequenced. The nucleotide sequence codes for a protein 397 amino acids including a putative signal peptide of 25 amino acids and a propeptide of 90 amino acids. The allergen is an alkaline serine protease that shares more than 39% identical residues with other kinds of mold allergens. The coding cDNA of Pen n 13 was cloned into vector pQE-30 and expressed in E. coli M15 as a His-tag fusion protein and purified to homogeneity. The fusion protein reacted with monoclonal antibodies of Pen c 1 and with IgE from Penicillium-allergic patients. Furthermore, it also cross-reacted strongly with IgE specific for the natural Pen c 1, indicating that similar IgE binding epitopes may exist in the allergens of P. notatum and P. citrinum. Antigenicity index plots indicated that there are several similar epitope regions of high antigenic indices in Pen c 1 and Pen n 13, corroborating that mold allergens belonging to the alkaline serine protease family possess similar protein structure and strong antigenic cross-reactivity.

A 10-year follow-up study of contraceptive Norplant implants.

OBJECTIVES: To evaluate the long-term efficacy and health problems associated with Norplant implants and re-implants after 5 full years of first implants. METHOD: From 1984 to 1988, 1657 women accepted first implants of Type I and Type II of Norplant, and 394 first acceptors had a re-implant, at a clinic for study. The clinical records and annual follow-up data of acceptors were analyzed. The life-table technique was the main method used. RESULTS: The continuation rates were very high. The cumulative pregnancy rates at 1, 3 and 5 full years of use were 0.0%, 0.1% and 0.7%, respectively. Users with heavier body weight had higher pregnancy rates. The rate of menstrual disturbances peaked at 73% after 3 months and consistently decreased to 20% at 5 years of use. Rates of menstrual disturbances associated with re-implants were much lower. CONCLUSION: Norplant is extremely effective and safe for long-term use.

Isolation and characterization of a novel proteinase inhibitor from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus).

A proteinase inhibitor (designated as TMI) was isolated and purified from the snake serum of Taiwan habu (Trimeresurus mucrosquamatus) by using successive chromatographies which included Sephadex G-100, DEAE-Sephacel chromatographies, and C(4) reverse-phase HPLC. The purified inhibitor was shown to be a homogeneous protein with a molecular mass of about 47 or 36 kDa in the presence or absence of a reducing agent, beta-mercaptoethanol. The inhibitor decreases in molecular mass by about 23% with N-linked neuraminidase treatment, suggesting that it is a glycoprotein. Further enzymatic analyses indicated that this inhibitor possesses strong inhibitory activities toward three zinc-dependent metalloproteinases and not fibrinogenolytic serine proteases previously isolated from the venom of the same snake species with an IC(50) of about 0.2-1.1 microM. Its IC(50) value was approximately three orders of magnitude more effective than those of the tripeptide inhibitors we previously purified from the crude venom of the same snake (Biochem. Biophys. Res. Commun. 248, 562-568 (1998)). The purified inhibitor showed stronger inhibitory action against caseinolytic activities of crude venoms from closely related species of Taiwan habu than those from unrelated species. N-terminal sequence analysis showed that its sequence is distinctly different from sequences of those serum inhibitors reported for other snake species in the literature. Based on inhibition susceptibility and primary structures of various snake protease inhibitors, it is suggested that this novel inhibitor isolated from the serum of Taiwan habu may be a unique self-defense protein factor mainly for protection against envenomation from snakes of the same genus.

Characterization of a novel allergen, a major IgE-binding protein from Aspergillus flavus, as an alkaline serine protease.

Aspergillus species of fungi have been known to be one of the most prevalent aeroallergens. One important A. flavus allergen (Asp fl 1) was identified by means of immunoblotting with a serum pool of allergic patients on a two-dimensional electrophoretic gel. The cDNA coding for Asp fl 1 was cloned and sequenced. The clone encodes a full-length protein of 403 amino acid precursors of 42 kDa. After cleavage of a putative signal peptide of 21 amino acids and a prepeptide of 100 amino acids, a mature protein of 282 amino acids was obtained with a molecular mass of 33 kDa and a pI of 6.3. A degree of identity was found in a range of 27 to 84% among related allergens derived from bacteria allergen subtilisin, mold allergen Pen c 1, and virulence factor of A. fumigatus. Recombinant Asp fl 1 (rAsp fl 1) was cloned into vector pQE-30 and expressed in E. coli M15 as a histidine-tag fusion protein and purified to homogeneity. The IgE binding capacity of rAsp fl 1 was tested by immunoblotting using a serum pool of Aspergillus-allergic patients. Recombinant allergen cross-reacted strongly with IgE specific for natural Asp fl 1 and Pen c 1, indicating that common IgE epitopes may exist between allergens of A. flavus and P. citrinum.

Identification and expression of Pen c 2, a novel allergen from Penicillium citrinum.

The mould genus, Penicillium, is known to be a significant source of environmental aero-allergens. One important allergen from Penicillium citrinum, Pen c 2, has been identified by means of two-dimensional immunoblotting using IgE-containing patients' sera. This novel allergen was cloned, sequenced and expressed in Escherichia coli. The cloned cDNA encodes a large 457-amino acid protein precursor containing a 16-amino acid signal peptide, a 120-amino acid propeptide and the 321-amino acid mature protein. Comparison of the Pen c 2 sequence with known protein sequences revealed shared high sequence similarities with two vacuolar serine proteases from Aspergillus niger and Saccharomyces cerevisiae. Asp-46, His-78 and Ser-244 were found to constitute the catalytic triad of the 39-kDa Pen c 2. The DNA coding for Pen c 2 was cloned into vector PQE-30 and expressed in E. coli as a His-tag fusion protein that bound serum IgE from Penicillium-allergic patients on immunoblots. Recombinant Pen c 2 could therefore be used effectively for diagnosis and also potentially for the treatment of mould-derived allergic disorders.