Biomedical subjects

L P Chow

Publications and source records attributed to L P Chow.

At least 19 recordsLinked to original sources

PubMed2005

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.

Adult↗

PubMed2001

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.

Abrin↗

PubMed2001

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.

Allergens↗

PubMed2001

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.

Acute Disease↗

PubMed2000

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.

Arsanilic Acid↗

PubMed2000

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.

Allergens↗

PubMed2000

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.

Allergens↗

PubMed2000

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.

Adult↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

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.

Allergens↗

PubMed1999

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.

Allergens↗

PubMed1999

Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina.

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inactivating protein (RIP), designated trichoanguin, which was purified to apparent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 of 6 microM. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N-glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, alpha-momorcharin, ricin A-chain and abrin A-chain reveals 55%, 48%, 36% and 34% identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and alpha-momorcharin. The large structural similarities might account for their common biological effects such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.

Amino Acid Sequence↗

PubMed1999

Pen c 1, a novel enzymic allergen protein from Penicillium citrinum. Purification, characterization, cloning and expression.

A 33-kDa alkaline serine protease secreted by Penicillium citrinum strain 52-5 is shown to be an allergenic agent in this fungus. The protein, designated Pen c 1, was purified by sequential DEAE-Sepharose and carboxymethyl (CM)-Sepharose chromatographies. Pen c 1 has a molecular mass of 33 kDa and a pI of 7.1. The caseinolytic enzyme activity of this protein was studied. The protein binds to serum IgE from patients allergic to Penicillium citrinum. The cDNA encoding Pen c 1 is 1420 bp in length and contains an open reading frame for a 397-amino-acid polypeptide. Pen c 1 codes for a larger precursor containing a signal peptide, a propeptide and the 33-kDa mature protein. Sequence comparison revealed that Pen c 1 possesses several features in common with the alkaline serine proteases of the subtilisin family. The essential Asp, His, and Ser residues that make up the catalytic triad of serine proteases are well conserved. Northern blots demonstrated that mRNAs transcribed from this gene are present at early stages of culture. The allergen encoded by Pen c 1 gene was expressed in Escherichia coli as a fusion protein bearing an N-terminal histidine-affinity tag. The protein, purified by affinity chromatography with a yield of 130 mg of pure protein per liter of culture, was able to bind to both a monoclonal anti-Pen c 1 antibody and IgE from the serum of patients allergic to Penicillium. Recombinant Pen c 1 can therefore be expressed in E. coli in large quantities and should prove useful as a standardized specific allergen for immuno-diagnosis of atopic disorders. In addition, full caseinolytic enzyme activity could be generated in the purified recombinant protein by sulfonation and renaturation, followed by removal of the affinity tag, indicating that the refolded protein can assume the same conformation as the native protein.

Allergens↗

PubMed1998

Sechiumin, a ribosome-inactivating protein from the edible gourd, Sechium edule Swartz--purification, characterization, molecular cloning and expression.

A new ribosome-inactivating protein (RIP), sechiumin, was purified from the seeds of edible gourd, Sechium edule Swartz by gel-filtration and ion-exchange chromatography, with an apparent relative molecular mass of 27 kDa. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50% inhibition (IC50) of 0.7 nM, but has a much lower inhibitory effect on intact HeLa cells, with an IC50 of 5000 nM. Sechiumin has a highly specific RNA N-glycosidase activity towards 28S rRNA, as does the A-chain of abrin. It suggests that sechiumin is one of the type-I ribosome-inactivating proteins. The cDNA of sechiumin has been cloned and expressed using a pET expression system in Escherichia coli. The sechiumin cDNA has 951 nucleotides, encoding a protein with 285 amino acids. The amino acid sequence deduced from the cDNA reveals that the first 21 N-terminal amino acid residues constitutes a signal peptide. Sechiumin has nearly 60% similarity to several type-I RIPs purified from the Cucurbitaceae family, such as luffin-a (62.5%) and trichosanthin (64.8%), but less similarity to other type-I RIPs. Two amino acid residues, Glu160 and Arg163, at the putative active site of sechiumin, are known to be catalytically active in ricin and abrin. The N-terminal amino acid sequence of sechiumin is very similar to that of trichosanthin. The recombinant sechiumin was obtained as an insoluble protein, and the preparation of the active soluble form was achieved by renaturing the denatured protein. These studies suggest that the recombinant sechiumin could be used for the preparation of immunotoxin as a potential cancer chemotherapeutic agent.

Abrin↗

PubMed1997

Bond-specific chemical cleavages of peptides and proteins with perfluoric acid vapors: novel peptide bond cleavages of glycyl-threonine, the amino side of serine residues and the carboxyl side of aspartic acid residues.

Peptide bond cleavages by vapors composed of various from aqueous solutions of perfluoric acid were studied using synthetic peptides and proteins, and specific conditions were established for peptide bond cleavages including a novel cleavage of the glycyl-threonine bond. The peptide bonds on the aminosides of serine residues were cleaved by exposure to a vapor of 75% aqueous heptafluorobutyric acid at 30 or 50 degrees C for 24 h. Glycyl-threonine peptide bonds were cleaved with vapors of various concentrations (5, 75, and 90%) of heptafluorobutyric acid at 30-40 degrees C for 24 h. The peptide bonds on the carboxylsides of aspartic acid residues were cleaved by exposure to a vapor of 0.2% heptafluorobutyric acid at 90 degrees C for 4 to 24 h. The same vapor cleaved aspartyl-proline bonds under milder conditions such as at 60 degrees C for 16 h, under which the other aspartyl bonds were uncleaved. These specific chemical cleavages were applied to several proteins including newly characterized proteins.

Amino Acid Sequence↗

PubMed1996

Amino acid sequence of the maize ferredoxin:thioredoxin reductase variable subunit.

The ferredoxin:thioredoxin reductase (FTR) is the essential enzyme of the light dependent regulatory system controlling enzyme activities in oxygenic, photosynthetic cells. This protein is composed of two dissimilar subunits, a catalytic subunit containing a [4Fe-4S] cluster and a redox-active disulfide bridge as the active site, and a variable subunit, whose function is not known yet. Whereas size and primary structure of the catalytic subunit from different organisms seem to be well conserved, they are quite variable for the variable subunit. Here we report the complete amino acid sequence of the variable subunit of maize (Zea mays) FTR established by protein sequencing. The subunit contains 97 residues and has a calculated molecular mass of 10939 Da. A sequence comparison shows 40% identity with the variable subunit from spinach and 38% with the one from Anacystis. The identical residues are grouped in three consensus domains, one near the N-terminus, one in the middle of the subunit and one near the C-terminus. We have obtained some evidence indicating that the N-terminal consensus domain is possibly involved in the interaction with the catalytic subunit.

Amino Acid Sequence↗

PubMed1996

Probing the domain structure of abrin-a by tryptic digestion.

Abrin-a is a potent plant toxin that consists of A and B chains linked by a disulfide bond. The abrin-a A chain (AaTA) has N-glycosidase activity while the abrin-a B chain (AaTB) has galactose-binding activity. By partial tryptic digestion, the domain structure of abrin-a was investigated. Seven tryptic fragments with molecular masses greater than 3500 Da were isolated and characterized. One fragment, designated T-21 and consisting of 153 amino acid residues, contained the major part of the second domain of AaTB and, after cross-linking of T-21 with glutaraldehyde, the reaction product had the same level of hemagglutinating activity as native abrin. When the T-21 fragment was conjugated with AaTA, the conjugate inhibited protein biosynthesis in HeLa cells. This suggests that the T-21 fragment is able to bind specifically to cells; its conjugate facilitates membrane translocation of AaTA into cells and consequently inhibits protein biosynthesis. T-21, with a molecular mass less than AaTB, is therefore a potentially useful substance for the preparation of immunotoxins.

Abrin↗

PubMed1996

Amino acid sequence of peptidyl-prolyl isomerase a of Fusarium sporotrichioides.

Peptidyl-prolyl-cis-trans isomerase catalyzes the interconversion of the cis and trans isomers of the proline-containing polypeptides and the folding process of proteins. This protein was known to be cyclophilin which has high binding affinity for cyclosporin A, a cyclic undecapeptide of fungal origin with potent immunosuppressive property agent. The two cytosolic peptidyl-prolyl-isomerases were found from Fusarium sporotrichioides. The amino acid sequence of the major peptidyl-prolyl isomerase a was determined by conventional sequencing methods; the protein with a calculated molecular mass of 19.7 kDa consisting of 179 amino acids. The comparison of the amino acid sequence of peptidyl-prolyl-isomerase from Fusarium with that of Nucerospora crassa revealed a significant degree of amino acid sequence homology (82.2%).

Amino Acid Isomerases↗