Yu Sam Kim

Humoral responses against the C-terminal region of merozoite surface protein 1 can be remembered for more than 30 years in persons exposed to Plasmodium vivax

Most people infected with Plasmodium vivax malaria developed antibodies against the C-terminal region of P. vivax merozoite surface protein (PvMSP1c) and the antibodies are sustained for a period up to 10xa0months after anti-malarial treatment. The longer-term stability of the specific humoral response was evaluated indirectly by determining the antibody titers in the sera from healthy individuals who lived an area from which malaria had been eradicated (450xa0persons) and an area in which it had recurred (1,524xa0persons). There were considerable residual antibody responses to PvMSP1c in over 15% of sera from healthy individuals, but only those who had lived in the era when malaria was prevalent. This means that antibodies against PvMSP1c may persist for more than 30xa0years, the malaria-free duration. This long-term memory of humoral immunity supports the C-terminal region of merozoite surface protein 1 as an effective malaria vaccine, in addition to the neutralizing activity reported previously.

A Proteomic approach for protein‐profiling the oncogenic ras induced transformation (H‐, K‐, and N‐Ras) in NIH/3T3 mouse embryonic fibroblasts

Point mutations in three kinds of Ras protein (H‐, K‐, and N‐Ras) that specifically occur in codons 12, 13, and 61 facilitate virtually all of the malignant phenotype of the cancer cells, including cellular proliferation, transformation, invasion, and metastasis. In order to elucidate an understanding into the oncogenic ras networks by H‐, K‐, and N‐Ras/G12V, we have established various oncogenic ras expressing NIH/3T3 mouse embryonic fibroblast clones using the tetracycline‐induction system, which are expressing Ras/G12V proteins under the tight control of expression by an antibiotics, doxycycline. Here we provide a catalog of proteome profiles in total cell lysates derived from three oncogenic ras expressing NIH/3T3 cells and a good in vitro model system for dissecting the protein networks due to these oncogenic Ras proteins. In this biological context, we compared total proteome changes by the combined methods of 2‐DE, quantitative image analysis, and MALDI‐TOF MS analysis using the unique Tet‐on inducible expression system. There were a large number of common targets for oncogenic ras, which were identified in all three cell lines and consisted of 204 proteins (61 in the pH range of 4–7, 63 in 4.5–5.5, and 80 in 5.5–6.7). Differentially regulated expression was further confirmed for some subsets of candidates by Western blot analysis using specific antibodies. Taken together, we implemented a 2‐DE‐based proteomics approach to the systematical analysis of the dysregulations in the cellular proteome of NIH/3T3 cells transformed by three kinds of oncogenic ras. Our results obtained and presented here show that the comparative analysis of proteome from oncogenic ras expressing cells has yielded interpretable data to elucidate the differential protein expression directly and/or indirectly, and contributed to evaluate the possibilities for physiological, and therapeutic targets. Further studies are in progress to elucidate the implications of these findings in the regulation of Ras induced transformation.

A direct sandwich ELISA to detect antibodies against the C-terminal region of merozoite surface protein 1 could be a useful diagnostic method to identify Plasmodium vivax exposed persons

Abstract. We expressed a C-terminal 108-aa region of the Plasmodium vivax merozoite surface protein 1 (PvMSP1c) excluding the C-end transmembrane region in Escherichia coli in order to evaluate the antibody level to MSP in Korean malaria patients. We optimized a direct sandwich enzyme-linked immunosorbent assay (ELISA) method to simultaneously determine the total antibody levels, including IgG and IgM, to PvMSP1c. If the cut-off for seropositivity was determined as the mean+3SD of the antibody levels of the negative control group, the antibody levels were positive in 99.5% of the patient group (sensitivity 199/200). The antibody levels were negative in 99.4% of the negative control group (specificity 504/507). The positive reactions in the negative control group came from non-specific reactions, as confirmed by a competition assay. This direct sandwich ELISA for PvMSP1c antibody could prove to be a useful tool for the diagnosis of malaria patients and for blood screening in blood banks.

Cloning and sequencing of genes encoding malonate decarboxylase in Acinetobacter calcoaceticus

Malonate decarboxylase from Acinetobacter calcoaceticus was isolated and characterized (Kim, Y.S., Byun, H.S., J. Biol. Chem. 269 (1994) 29636-29641), and its subunits were reanalyzed recently to be alpha, beta, gamma, and delta. The genes for the subunits, MdcA (548 a.a.), B (295 a.a.), C (238 a.a.), and D (102 a.a.), of the enzyme have been cloned by using oligonucleotide primers deduced from amino acid sequences of peptides isolated from the purified enzyme, and sequenced to be clustered in an operon in the order of A-D-B-C. The operon was found to encode more genes than mdcABCD. The Escherichia coli, transformed with the vector containing the insert mdcADBC and about 1.7 kb of an upstream region, expressed the four subunits of the enzyme but the proteins did not show enzyme activity. It indicates that, like the enzymes from Malonomonas rubra and Klebsiella pneumoniae, more genes are needed for the formation of the functional malonate decarboxylase.

Diagnosis of vivax malaria using an IgM capture ELISA is a sensitive method, even for low levels of parasitemia

Although diagnosis of Plasmodium vivax malaria has been difficult when it is present at a low parasite density, it was recently revealed that an antibody assay was a good method of screening for malaria in blood banks. However, the use of this method for the diagnosis of malaria is limited due to the persistence of specific immunoglobulin (Ig) G. Therefore, we evaluated specific IgM antibody responses against the C-terminal region of the merozoite surface protein 1 of P. vivax (PvMSP1c) in sera obtained from patients with vivax malaria using various assays. The IgM capture enzyme-linked immunosorbent assay showed good sensitivity (97.7%; 308/315) and specificity (99.1%, 446/450). In addition, the results of this assay were not related to parasite density, and a high reactivity was observed when there was a low level of parasitemia. Furthermore, we found that patients with cases of malaria that had relapsed still had the IgM titers against PvMSP1c. Therefore, the use of IgM ELISA for the detection of specific IgM that was not involved in memorial immune activity could be an alternative tool for the diagnosis of malaria and blood screening, even in areas in which malaria is endemic.

Genomic organization and characterization of the promoter of rat malonyl-CoA decarboxylase gene

Malonyl-CoA decarboxylase (MCD) catalyzes the decarboxylation of malonyl-CoA, an elongating agent for fatty acid synthesis and also known as a fuel-sensing mediator. In order to elucidate the genome organization, we isolated a 2020 bp rat MCD cDNA from rat brain cDNA library and isolated the corresponding rat genomic clones from the rat genomic PAC library. Sequencing and comparison of these clones showed that the MCD genome consists of five exons and four introns spanning approximately 17 kb. The proximal upstream region is GC-rich, lacks a TATA box, and contains a variety of putative transcriptional regulatory elements within 2 kb. A major transcriptional initiation site was identified by a primer extension at a site 157 nucleotides upstream of the translational initiation site. To investigate the transcriptional regulation of MCD, a series of 5-deletion constructs of the 5-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in CV-1 cells, we suggest that an area of -15 bp 5 from the first exon acted as a basal promoter for MCD and that there are positive cis-regulatory elements in the region from -55 to -325 bp and negative regulator elements in the region -1380 to -2240 bp.

Inactivation of Acinetobacter calcoaceticus acetate kinase by diethylpyrocarbonate

Acetate kinase purified from Acinetobacter calcoaceticus was inhibited by diethylpyrocarbonate with a second-order rate constant of 620 M-1.min-1 at pH 7.4 at 30 degrees C and showed a concomitant increase in absorbance at 240 nm due to the formation of N-carbethoxyhistidyl derivative. Activity could be restored by hydroxylamine and the pH curve of inactivation indicates the involvement of a residue with a pKa of 6.64. Complete inactivation of acetate kinase required the modification of seven residues per molecule of enzyme. Statistical analysis showed that among the seven modifiable residues, only one is essential for activity. 5,5-dithiobis(2-nitrobenzoic acid), p-chloromercuryphenylsulfonate, N-ethylmaleimide and phenylglyoxal did not affect the enzyme activity. These results suggest that the inactivation is due to the modification of one histidine residue. The substrates, acetate and ATP, protected the enzyme against inactivation, indicating that the modified histidine residue is located at or near the active site.