Ribozymes are catalytic RNAs that can cleave RNAs at specific sites, thus they have been employed to degrade a target mRNA in vivo. Development of allosterically controllable ribozymes is of great current interest, but it remained difficult to furnish such functions to ribozymes in cultured cells or in animals. Recently, we designed allosterically controllable ribozymes termed maxizymes, which have sensor arms that recognize target mRNA sequences and, in the presence of such target sequences only, they form a cavity that can capture catalytically indispensable $Mg^{2+}$ ions, cleaving the target. The maxizyme was applied to therapy for chronic myelogenous leukemia (CML). It cleaved specifically the chimeric BCR-ABL mRNA, which caused CML, without damaging the normal ABL or BCR mRNA in mammalian cells and also in mice, providing the first successful example for allosteric control of the activity of artificial ribozymes in vivo.

The nucleotide effects of Hafnia alvei aspartase were investigated. Purine nucleosides, such as adenosine and guanosine, increased the aspartase activities; whereas, purine nucleotides, such as AMP, ATP, GTP and IMP, caused little change in the aspartase activities. However, pyrimidine derivatives, such as cytidine and CTP, decreased the aspartase activity. The nucleotide and nucleoside effects by the limited trypsin-treated aspartase were similar to those of a native enzyme. These results indicate that the COOH-terminal region and an allosteric site might be located away from each other. The initial velocity study in the presence of adenosine showed that $K_m$ for aspartate was decreased to one-sixth of that in the absence of adenosine, but $V_{max}$ was unchanged. The significance of the distinct allosteric effect for the enzyme-nucleotide interaction is discussed.

Incubation of purified laminin1-nidogen1 complexes with $[{\gamma}-^{32}P]-ATP$ in the presence of the catalytic subunit of the protein kinase A (cAMP-dependent protein kinase) resulted in the phosphorylation of the alpha chain of laminin-1 and of the nidogen-1 molecule. Aminoacid electrophoresis indicated that phosphate was incorporated on serine residues. The phosphorylation effect of laminin-1 on the process of self assembly was studied by turbidometry. In these experiments, the phosphorylated laminin-1 showed a reduced maximal aggregation capacity in comparison to the non-phosphorylated molecule. Examination of the laminin-1 network under the electron microscope showed that the phosphorylated sample formed mainly linear extended oligomers, in contrast to controls that formed large and dense multimeric aggregates. Heparin binding on phosphorylated laminin-1 in comparison to controls was also tested using solid-phase binding assays. The results indicated an enhanced heparin binding to the phosphorylated protein. The results of this study indicate that laminin1-nidogen1 is a substrate for protein kinase A in vitro. This phosphorylation had an obvious influence on the lamininl-nidogen1 network formation and the heparin binding capacity of this molecule. However, further studies are needed to investigate whether or not this phenomenon could play a role in the formation of the structure of basement membranes in vivo.

The native conformation of serpins (serine protease inhibitors) is strained. Upon cleavage of the reactive center loop of serpins by a protease, the amino terminal portion of the cleaved loop is inserted into the central ${\beta}-sheet$, A sheet, as the fourth strand, with the concomitant release of the native strain. We questioned the role of protease in this conformational switch from the strained native form into a stable relaxed state. Chemical cleavage of the reactive center loop of ${\alpha}_1-antitrypsin$, a prototype serpin, using hydroxylamine dramatically increased the stability of the serpin. A circular dichroism spectrum and peptide binding study suggests that the amino terminal portion of the reactive center loop is inserted into the A sheet in the chemically-cleaved ${\alpha}_1-antitrypsin$, as in the enzymatically-cleaved molecule. These results indicate that the structural transformation of a serpin molecule does not require interaction with a protease. The results suggest that the serpin conformational switch that occurred during the complex formation with a target protease is induced by the cleavage of the reactive center loop per se.

Overwintering plants should survive the various biotic and abiotic stresses that occur during winter. Previous studies indicated that active oxygen species are involved in freezing, dehydration, anoxia and pathogen infections. As the importance of the events that occur in the intercellular compartment became apparent in disease resistance, we examined the nature of intercellular antioxidant enzymes in order to access their possible involvement in the winter hardiness of barley. The levels of intercellular peroxidase, catalase, and SOD activities on the unit protein basis were 394, 18, and 9% of those of cellular activities, respectively. Major intercellular peroxidase isoforms consisted of four neutrals and four basic forms; whereas major cellular isoforms were two basic forms. Out of the two major catalase isoforms a higher molecular weight form was predominantly abundant in both cellular and intercellular compartments. Among the five major cellular SOD isoforms, three were also present in the intercellular compartment. The presence of substantial amounts of intercellular antioxidant enzymes in overwintering barley leaves may suggest the involvement of these enzymes in the tolerance mechanism to the various stresses that occur during winter.

Calexcitin, a calcium-binding protein, was previously cloned and functionally characterized in the squid Loligo pealei. We now report the cloning of a second form of Calexcitin, Calexcitin-2, found in the squid Todarodes pacificus optic lobe. Calexcitin-2 has a significantly different carboxyl terminal region than Calexcitin-1. It lacks the CAAX motif, which is a farnesylation site. The amino acid sequence of Calexcitin-2 shows an 84% identity with Calexcitin-1 and also displays a strong cross immunoreactivity. Western blotting shows that Calexcitin-2 was expressed exclusively in the optic lobe region of squid, but not in other body organs. Regardless of its lack of conserved regions for GTP-binding, Calexcitin-2 shows moderately low affinity GTP-binding and also shows dramatic conformational change induced by GTP-binding. Three possible GTP-binding region mutations, K142A, D144A, and K157A, did not change the G TP binding affinity. This raises the possibility that Calexcitin-2 may have a novel GTP-binding motif.

A differential display method is used to identify novel genes whose expression is affected by treatment with ferric ammonium citrate (FAC) or desferrioxamine (DFO), an iron chelating agent in the human hepatoblastoma cell line (HepG2). These chemicals are known to deplete or increase the intracellular concentration of iron, respectively. Initially, we isolated seventeen genes whose expressions are down- or up regulated by the treatment of the chemicals, as well as their four differentially expressed genes that are designated as clone-1, -2, -3, and -4. These are further characterized by cDNA sequencing and Northern blot analysis. Through the cDNA sequencing, as well as comparing them to genes published using the NCBI BLAST program, we identified the sequence of the clone-1 that is up-regulated by the treatment of DFO. It is identical to the human insulin-like growth factor binding protein-1 (IGFBP-1). This suggests that the IGFBP-1 gene in the HepG2 cell is up-regulated by an iron depletion condition. Also, the expression of the clone-3 and -4 is up-regulated by FAC treatment and their eDNA sequences are identical to the human ferritin-fight chain and human NADH-dehydrogenase, respectively. However, the sequence of the clone-2 has no significant homology to any other known gene. Therefore, we suggest that changes of the cellular iron level in the HepG2 cell affects the transcription of cellular genes. This includes human IGFBP-1, ferritin-fight chain, and NADH-dehydrogenase. Regulation of these gene expressions may have an important role in cellular functions that are related to cellular iron metabolism.

In order to understand the role of AGP on the differentiation of promyelocytic leukemia cells, the AGP expression and its relation to cytokines were investigated during granulocytic or monocytic differentiation of HL-60 cells. When HL-60 cells were treated with all-trans-retinoic acid (ATRA) for 5 days, the cells were fully differentiated into granulocytes, and the AGP mRNA and protein levels were continuously increased up to 5 days in a dose- and time- dependent manner. However, in the case of the monocytic differentiation of HL-60 cells by tetradeanoyl phorbol acetate (TPA), the AGP gene expression was not induced. In addition, $IL-1{\alpha}$, $IL-1{\beta}$, IL-6 and $TNF-{\alpha}$ mRNAs were also enhanced during granulocytic differentiation. These cytokine transcripts showed a peak level 3 days after the ATRA treatment. It decreased gradually thereafter. However, direct addition of recombinant cytokines ($IL-1{\beta}$, IL-6 and $TNF-{\alpha}$) and dexamethasone to the HL-60 cell cultures showed no AGP induction. These findings suggest that the AGP and proinflammatory cytokines are expressed in ATRA-treated promyelocytic cells. However, these cytokines do not act as autocrine inducers on AGP expression. This fact implies that the AGP expression during granulocytic differentiation of HL-60 cells is induced through a signal pathway different from hepatocyte signaling in inflammation.

The effects of heat shock, or all-trans retinoic acid, on the expression of the C-reactive protein mRNA in the HT-29 human colon carcinoma cells, as well as the functional role of the C-reactive protein as a molecular chaperone, were studied. The expression level of the C-reactive protein mRNA in the HT-29 cells was increased time-dependently when exposed to heat-shock, and dose-dependently when treated with all-trans retinoic acid. The activities of transglutaminase C and K in the HT-29 cells were significantly increased when treated with all-trans retinoic acid. The C-reactive protein prevented thermal aggregation of the citrate synthase and stabilized the target enzyme, citrate synthase. The C-reactive protein promoted functional refolding of the urea-denatured citrate synthase up to 40-70%. These results suggest that the C-reactive protein, which is induced in human colon carcinoma cells, when heated or treated with all-trans retinoic acid has in a part functional activity of the molecular chaperone.

The obligate aerobic Deinococcus are highly resistant against lethal effect of UV-and ionizing-radiation. Only five mesophilic Deinococcus species, i. e. D. radiodurans, D. radiophilus, D. proteolyticus, D. radiopugnans, and D. grandis are known. Since an indispensable role of catalase has been suggested in protecting cells against oxidative stress and UV radiation, Deinococcal catalase activity of each species and electrophoretic profiles of catalases were investigated on gel. Total catalase activity was varied among the species in the aerobically grown culture at stationary phase. The occurrence of multiple forms of catalases with different molecular weights in four species of Deinococcus and of a single catalase in D. radiopugnans suggests that each species shows the unique catalase profiles on gel. Some Deinococcal catalases also exhibit peroxidase activity. Since Deinococcus spp. are less-distinct to each other in their morphology, biochemical and physiological properties, the catalase profiles on PAGE would be useful in identifying the species of Deinococcus.

Mitochondrial DNA (mtDNA) mutation was investigated in a hepatoma patient using a polymerase chain reaction (PCR) and an in situ hybridization technique. Biotin-labeled probes for the subunit m of cytochrome c oxidase revealed differences in the in situ hybridization. A PCR assay using biopsied and microdissected tissues showed that common deletion (4,977 bp) was more pronounced in the cancer region than in the normal parts of the same patient. These results suggest that mtDNA deletion might be associated with tumorigenesis in hepatoma.

Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme catalyzing the reduction of a variety of disulfide compounds and acting as a cofactor for various enzymes such as ribonucleotide reductase. The Schizosaccharomyces pombe cells, exponentially grown in rich medium at $30^{\circ}C$, were shifted to $20^{\circ}C$ and $35^{\circ}C$. The yeast cells, shifted to $35^{\circ}C$, showed higher TTase activity than the cells continuously grown at $30^{\circ}C$, whereas the yeast cells, shifted to $20^{\circ}C$, gave lower TTase activity. The S. pombe cells, exponentially grown in minimal medium and shifted from $30^{\circ}C$ to $35^{\circ}C$ and $40^{\circ}C$, produced higher TTase activity. When the S. pombe cells were initially incubated in rich and minimal media at three different temperatures ($25^{\circ}C$, $30^{\circ}C$ and $35^{\circ}C$), they showed higher TTase activity at higher temperature. These results suggest that the TTase activity of S. pombe is regulated by temperature.