Jongweon Lee

Molecular cloning and characterization of a major egg antigen in Paragonimus westermani and its use in ELISA for the immunodiagnosis of paragonimiasis

A recombinant protein of a Paragonimus westermani egg antigen was produced and tested as an antigen for the serologic diagnosis of P. westermani infection. The P. westermani egg antigen gene contains a single open reading frame of 966 base pairs encoding 322 amino acids from 5′ methionine to the 3′ stop codon. The predicted amino acid sequence of this egg antigen was 40, 38, and was 35% identical to heat shock proteins from Schistosoma japonicum, Schistosoma mansoni, and Taenia saginata. The distribution this antigen was investigated in adult worms by indirect immunofluorescence assay, and found to be distributed in eggs and uteri. The specificity and sensitivity of the recombinant antigen were assessed by enzyme-linked immunosorbent assay (ELISA) using sera from patients infected with different parasites, which included 41 patients with paragonimiasis, and negative controls. The diagnostic positive and negative predictive absorbance value was 0.24 and the sensitivity of ELISA using the recombinant antigen was 90.2%, and its specificity 100%. Our results suggest that the developed recombinant major egg antigen-based ELISA offers a highly sensitive and specific assay for the diagnosis of paragonimiasis.

Immunoglobulin E Reactivity of Recombinant Allergen Tyr p 13 from Tyrophagus putrescentiae Homologous to Fatty Acid Binding Protein

ABSTRACT The storage mite, Tyrophagus putrescentiae, is one of the important causes of allergic disorders. Fifteen allergenic components were demonstrated in storage mite by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, but only the group 2 allergen Tyr p 2 has been cloned and characterized. In this study, we attempted to identify and characterize new allergens from T. putrescentiae, which is a dominant species of storage mite in Korea. Expressed sequence tags were analyzed to identify possible storage mite allergens, and the cDNA sequence encoding a protein homologous to fatty acid binding protein, a mite group 13 allergen, was identified and named Tyr p 13. Its deduced amino acid sequence showed 61.1 to 85.3% identity with other mite group 13 allergens. The recombinant protein was expressed in Escherichia coli using a pET 28b vector system, and its allergenicity was investigated by enzyme-linked immunosorbent assay (ELISA). The recombinant allergen was detected in 5 of 78 (6.4%) T. putrescentiae-positive sera tested, and it inhibited 61.9% of immunoglobulin E binding to crude extract at an inhibitor concentration of 10 μg/ml by inhibition ELISA using serum from the patient who showed the strongest reaction by ELISA. In this study, a novel allergen was identified in T. putrescentiae. This allergen could be helpful for more-detailed characterizations of storage mite allergy.

Molecular Cloning and the Allergenic Characterization of Tropomyosin from Tyrophagus putrescentiae

Storage mites have been recognized as a cause of asthma and rhinitis. Studies from several countries have shown that the IgE-mediated allergy to storage mites is of considerable importance, especially in rural populations. This study aimed to identify and characterize new allergens from Tyrophagus putrescentiae. A partial cDNA sequence encoding tropomyosin was isolated from the cDNA library by immunoscreening using anti-mouse IgG1 sera raised against T. putrescentiae whole body extract. The deduced amino acid sequence shares 64-94% identity with previously known allergenic tropomyosins. Its recombinant protein was produced by using a pET 28b expression system and purified by affinity chromatography using Ni-NTA agarose. The IgE reactivities of tropomyosins from T. putrescentiae and Dermatophagoides farinae were compared by enzyme linked immunosorbent assay (ELISA). Recombinant Tyr p 10 showed 12.5% (5/40) IgE-binding reactivity, whereas recombinant Der f 10 showed 25% (10/40) IgE-binding reactivity against the same sera from storage mite-sensitized and house dust mite-sensitized subjects. Both recombinant Tyr p 10 and Der f 10 showed little inhibition of IgE binding to T. putrescentiae crude extract by ELISA. Tropomyosin seems to contribute only a small portion of the cross-reactivity with house dust mites.

Effect of Iron on Adherence and Cytotoxicity of Entamoeba histolytica to CHO Cell Monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.

Allergenic Characterization of Tropomyosin from the Dusky Brown Cockroach, Periplaneta fuliginosa

ABSTRACT Household arthropods are one of the most common causes of allergic diseases. Four species of cockroaches are found to reside in Korean homes, but published work deals almost exclusively with the German and American cockroaches. This study was undertaken to investigate the cross-reactive allergenic components of the dusky brown cockroach, Periplaneta fuliginosa. Enzyme-linked immunosorbent assay (ELISA) inhibition and immunoblot analyses for the dusky brown cockroach were performed with Blattella germanica and Dermatophagoides farinae allergic sera. cDNA encoding tropomyosin, which is a well known cross-reactive pan-allergen, was cloned by reverse transcriptase PCR, and recombinant protein was produced by using a pET-28b expression system. Native tropomyosin was purified by ammonium sulfate fractionation and electroelution. The immunoglobulin E (IgE) reactivities of native and recombinant tropomyosins were compared by an ELISA inhibition study. All 30 sera tested showed P. fuliginosa-specific IgE, and the IgE-binding reactivity of the P. fuliginosa extract was inhibited as much as 79.4% by a B. germanica extract and as much as 63.3% by a D. farinae extract. The deduced amino acid sequence of cloned cDNA was identical with that of Periplaneta americana tropomyosin (98.5% nucleotide sequence identity). Seven of 26 (26.9%) allergic sera had IgE specific for recombinant protein, and the maximum inhibition of P. fuliginosa-specific IgE achieved with recombinant tropomyosin was 37.7% at an inhibitor concentration of 10 μg/ml. Native tropomyosin inhibited the binding of IgE to the P. fuliginosa, B. germanica, and D. farinae extracts by 65.0, 51.8, and 39% at an inhibitor concentration of 1 μg/ml. P. fuliginosa appears to possess allergens that are highly cross-reactive with allergens of B. germanica and D. farinae. Tropomyosin was found to be a major allergenic component accounting for the cross-reactivity between cockroaches and dust mites.

Immunoglobulin E Binding Reactivity of a Recombinant Allergen Homologous to α-Tubulin from Tyrophagus putrescentiae.

ABSTRACT Storage mites may cause allergic respiratory diseases in urban areas as well as pose an occupational hazard in rural areas. Characterization of storage mite allergens is important for the development of diagnostic and therapeutic agents against mite-associated allergic disorders. Here we report on the cloning and expression of α-tubulin from the storage mite (Tyrophagus putrescentiae). The deduced amino acid sequence of the α-tubulin from the storage mite showed as much as 97.3% identity to the α-tubulin sequences from other organisms. The highly conserved amino acid sequences of α-tubulins across different species of mites may indicate that cross-reactivity for this potential allergen exists. The frequency of immunoglobulin E reactivity of this recombinant protein is 29.3% in sera from storage mite-allergic subjects.

Population Dynamics of Five Anopheles Species of the Hyrcanus Group in Northern Gyeonggi-do, Korea

To investigate the population densities of potential malaria vectors, Anopheles species were collected by light traps in malaria endemic areas, Paju and Gimpo, Gyeonggi-do of Korea. Five Anopheles Hyrcanus sibling species (An. sinensis, An. pullus, An. lesteri, An. kleini, and An. belenrae) were identified by PCR. The predominant species, An. pullus was collected during the late spring and mid-summer, while higher population consists of An. sinensis were collected from late summer to early autumn. These 2 species accounted for 92.1% of all Anopheles mosquitoes collected, while the other 3 species accounted for 7.9%. Taking into account of these population densities, late seasonal prevalence, and long-term incubation period (9-13 months) of the Korean Plasmodium vivax strain, An. sinensis s.s is thought to play an important role in the transmission of vivax malaria in the study areas.

Analysis of Amino Acid Sequence Variations and Immunoglobulin E-Binding Epitopes of German Cockroach Tropomyosin

ABSTRACT The allergenicities of tropomyosins from different organisms have been reported to vary. The cDNA encoding German cockroach tropomyosin (Bla g 7) was isolated, expressed, and characterized previously. In the present study, the amino acid sequence variations in German cockroach tropomyosin were analyzed in order to investigate its influence on allergenicity. We also undertook the identification of immunodominant peptides containing immunoglobulin E (IgE) epitopes which may facilitate the development of diagnostic and immunotherapeutic strategies based on the recombinant proteins. Two-dimensional gel electrophoresis and immunoblot analysis with mouse anti-recombinant German cockroach tropomyosin serum was performed to investigate the isoforms at the protein level. Reverse transcriptase PCR (RT-PCR) was applied to examine the sequence diversity. Eleven different variants of the deduced amino acid sequences were identified by RT-PCR. German cockroach tropomyosin has only minor sequence variations that did not seem to affect its allergenicity significantly. These results support the molecular basis underlying the cross-reactivities of arthropod tropomyosins. Recombinant fragments were also generated by PCR, and IgE-binding epitopes were assessed by enzyme-linked immunosorbent assay. Sera from seven patients revealed heterogeneous IgE-binding responses. This study demonstrates multiple IgE-binding epitope regions in a single molecule, suggesting that full-length tropomyosin should be used for the development of diagnostic and therapeutic reagents.

PRELIMINARY OBSERVATION ON THE GRADIENT DISTRIBUTION OF HIBERNATING FEMALES OF ANOPHELES SINENSIS AND ANOPHELES PULLUS IN THE REPUBLIC OF KOREA

ABSTRACT Hibernating females of the Anopheles hyrcanus complex were collected at 4 different latitudes at the end of the hibernation period in March of 2005 and 2006, and species were confirmed by polymerase chain reaction (PCR) identification. At Paju (37.8°N; average temperature in December–February  =  −3.5°C) 74.6% of the females collected were An. pullus and 22.4% were An. sinensis. At Seosan (36.8°N; December–February average temperature  =  −1.0°C) and Buan (35.7°N; December–February average temperature  =  0.4°C) 14.7% and 14.8% were An. pullus, and 85.3% and 85.2% were An. sinensis, respectively. At Haenam (34.6°N; December–February average temperature  =  3.1°C) only An. sinensis was found. Our preliminary results indicate that An. sinensis, unlike An. pullus, is not adapted to survive in cold weather.

Regulation of two soluble forms of brain glutamate dehydrogenase isoproteins by protein kinases

We isolated two soluble forms of glutamate dehydrogenase isoproteins, GDH I and GDH II, from bovine brain. The regulation of GDH I and GDH II by phosphorylation and dephosphorylation has been examined in various conditions. There were dose‐ and time‐ dependent activation of the GDH isoproteins when phosphorylated by cAMP‐dependent protein kinase. The phosphorylated GDH had 1.1 mol of covalently bound phosphate/mol of subunit and a 2‐fold increased specific activity. The phosphorylated amino acid was identified as serine. When treated with alkaline phosphatase, the activities of the phosphorylated GDH isoproteins were reduced in dose and time dependent manner and returned to those of unphosphorylated enzymes. There were no significant differences between GDH I and GDH II in their sensitivities to the action of phosphorylation and dephosphorylation, demonstrating that the microenvironmental structures of the phosphorylation site in GDH isoproteins are similar to each other. These results suggest that the in...