Modification of the inhibitory amino acid for epitope peptide binding onto major histocompatibility complex class II molecules enhances immunogenicity of the antigen.

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.

Enhanced expression and functional characterization of the human ferritin H- and L-chain genes in Saccharomyces cerevisiae.

Enhanced expression of the human ferritin H- and L-chain genes (hfH and hfL) was achieved in Saccharomyces cerevisiae by modifying the N-terminal region of the structural genes. The yeast episomal vector YEp352 with the galactokinase1 (GAL1) promoter was used to construct expression plasmids. The expression of each gene was examined using SDS-PAGE and Western blot analysis. Iron uptake was examined and the cellular iron concentration was increased in S. cerevisiae expressing hfH. When cultured cells were incubated with 14.3 mM Fe(2+), the recombinant yeast expressing hfH had a cellular iron concentration 1.5 times greater than that of the control strain. The relationship between the iron taken up by the cells and the expressed proteins was examined. Iron-binding H-chain ferritin (H-ferritin) was seen in the recombinant S. cerevisiae incubated with iron, while small amounts of iron-binding L-chain ferritin (L-ferritin) were observed. Combined, these observations demonstrate that human H-ferritin has a function in iron storage in S. cerevisiae, while L-ferritin does not.

Amplified CDK2 and cdc2 activities in primary colorectal carcinoma.

BACKGROUND: Cyclins are overexpressed in various malignancies, including carcinoma of the colorectum, esophagus, lung, larynx, and breast. However, to the authors' knowledge, the protein levels and activities of cyclin-dependent kinases (CDKs), which are the functional cyclin partners in the cell cycle, have not been investigated previously. METHODS: Eight samples of cancer tissue and adjacent normal tissue were taken from 23 patients with Stage B2-C1 (AJCC/UICC Stage II-III) colorectal carcinoma during curative resection. The protein levels of cyclin and CDKs were determined by Western blot analysis. The activities of CDKs were determined by the phosphorylation amount using specific substrates after immunoprecipitations. RESULTS: The protein expression of cyclin (D1, D3, E, and A) and CDKs (CDK4, CDK2, and cdc2) was higher in primary colorectal carcinoma tissue than in adjacent normal tissue. Whereas only 3 of 8 patients had increased CDK4 activity in cancer tissue, 8 of 8 and 7 of 8 patients had increased CDK2 and cdc2 activities, respectively, in cancer tissue compared with adjacent normal tissue. However, there were no positive correlations among the pathologic staging or differentiation status and the increased ratio of cyclin protein, CDK protein, or CDK activity. CONCLUSIONS: These results indicate that significant activation of S and M phases of the cell cycle occurs in primary colorectal carcinoma.

Economic miracle for what?: State workers' health in South Korea.

This article is a study of the state's role in workers health in South Korea during the period of the 1950s to 1980s in which South Korea achieved its economic success through a series of economic development plans. The state's role in the protection of workers health will be examined by investigating the historical development of two main welfare state programs, workers' compensation and national health insurance, as the pillars of state policies on workers health. In contrast to the state's direct intervention in economic development, I will argue, the key characteristics of both workers' compensation and national health insurance are the state's minimal organizational and financial costs and the relative autonomy of firm managers. Also, the state first restricted the scope of beneficiaries to the core group of manufacturing and mining workers and then gradually expanded it over a long period of time. I will argue that such features suggest a strong dependence on business by the Korean welfare state programs that contradicts the image of a strong state that the scholars of East Asian states often claim.

The Brevibacterium albidum gene encoding the arginine tRNACCG complements the growth defect of an Escherichia coli strain carrying a thermosensitive mutation in the rnpA gene at the nonpermissive temperature.

The Escherichia coli rnpA gene encodes C5 protein, the protein component of RNase P. The rnpA49 mutation renders the C5 protein thermosensitive, which results in thermosensitivity of RNase P function. The chromosomal DNA region from Brevibacterium albidum that complements the rnpA49 mutation was analysed. The gene capable of complementing the growth defect of an rnpA49 mutant strain at nonpermissive temperature was identified as the gene for an arginine tRNA with anticodon CCG by a deletion analysis combined with complementation assays. Transcription of the arginine tRNA gene carried on a multicopy plasmid was correlated with the complementation of the rnpA49 mutation, indicating that the gene product is indeed responsible for complementation of the rnpA49 mutation.

Selectivity of phospholipase C isozymes in growth factor signaling.

Xenopus laevis oocytes were injected with mRNA extracted from growth factor-responsive CCL39, Chinese hamster lung fibroblasts. The expression of functional growth factor receptors on the oocytes was demonstrated by growth factor-induced 45Ca2+ efflux. To determine the isozyme(s) of phospholipase C (PLC) coupled to growth factor receptors, growth factor-induced 45Ca2+ efflux were measured following coinjection of mRNA from CCL39 cells with PLC antibodies. PLC-gamma 1 antibody did not lead to loss of 45Ca2+ efflux induced by thrombin but resulted in loss of that induced by platelet-derived growth factor (PDGF). In contrast, PLC-delta 1 antibody did not block PDGF-induced 45Ca2+ efflux but led to inhibition of thrombin-induced 45Ca2+ efflux. PLC-beta 1 antibody did not affect Ca2+ efflux by the treatment of either thrombin or PDGF. These results suggest that these growth factor receptors are coupled to specific effectors, i.e. thrombin receptor to PLC-delta 1 and PDGF receptor to PLC-gamma 1.

Single injection of pentobarbital induces long-lasting effects on ANP synthesis and gene expression in the rat atria.

To define the long-term effects of pentobarbital sodium on the plasma and atrial atrial natriuretic peptide (ANP) system, experiments were performed in female Sprague-Dawley rats. The plasma levels of immunoreactive (ir) ANP showed chronic as well as acute response to pentobarbital sodium administration. A single dose (30 mg/kg, i.p.) of pentobarbital sodium resulted in a suppression in the plasma levels of irANP up to 1 week of administration. The suppressive effect on plasma irANP concentrations was dose-dependent. Right but not left atrial contents of irANP increased by an administration of pentobarbital sodium up to 4 weeks. ANP mRNA contents of the atrial exposed to pentobarbital sodium began to increase after 2 days, reached to the peak after 2 weeks, and began to return to control values after 6 weeks. Surgical stress accentuated these patterns of plasma and atrial ANP responses to pentobarbital sodium treatment. The present data, therefore, suggest that even a single anesthetic dose of pentobarbital could elicit long-lasting profound changes in ANP system, i.e., changes in gene expression, synthesis and the secretion of ANP.

Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript.

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.