A number of anticancer-chemotherapeutic agents induce cell death through the process of apoptosis. Effects of echinomycin, an anticancer agent on cancer progression, were investigated in P388 murine leukemia cells. First, according to the results of cytotoxicity measurement. $IC_{50}$ of echinomycin was 1.12 nM, a relatively lower value than the other examined anticancer agents, mitomycin-C and etoposide Second, the DNA fragmentation assay for echinomycin-treated cells exhibited that echinomycin was able to induce apoptosis in a shorter period of time and with a lower dose than mitomycin-C or etoposide. The data of DNA fragmentation were quite comparable to those of cytotoxicity measurement. Finally we showed that mitogen-activated protein (MAP) kinase, a key protein in cell mitosis, was translocated into the nucleus from the cytosol after treatment with echinomycin. These findings suggest that a MAP kinase-related process may be involved in apoptosis induced by echinomycin.

The biochemical properties of purified D-hydruxyisovalerate dehydrogenase from Fusarium sambucinum was elucidated. D-Hydroxyisovalerate dehydrogenase produced solely D-hydroxyisovalerate from 2-ketoisovalerate. The isoelectric point of the purified enzyme was 7.0. The enzyme was highly specific with 2-ketoisovalerate ($K_{m}=0.188$ mM, $V_{max}=8.814$ mmol/min mg) and 2-keto-3-methyl-n-valerate ($K_{m}=0.4$ mM, $V_{max}=1.851$ mmol/min mg) for the reductive reaction. This was also seen by comparing D-hydroxyisovalerate ($K_{m}=1.667$ mM, $V_{max}=0.407$ mmol/min mg) and D-hydroxy-3-methyl-n-valerate ($K_{m}=6.7$ mM, $V_{max}=0.648$ mmol/min mg) for the oxidative reaction. Thiol blocking reagents, such as iodoacetamide, N-ethylmaleimide and p-chloromecuribenzoate inhibited about 80% of enzyme activity at 0.02 mM, 50 mM and 50 mM, respectively. The enzyme activity was also inhibited by the addition of 0.1 mM of various metal ions, such as $Fe^{2+}$ (67%), $Cu^{2+}$ (88%), $Zn^{2+}$ t (76%) and $Mg^{2+}$ (9%). The enzyme was stable over three months in 50 mM potassium phosphate buffer (pH 5~7) at $-80^{\circ}C$. However the purified enzyme lost 30% of its activity in the same buffer after 24 h at $4^{\circ}C$. The studies about thermal inactivation of D-hydroxyisovalerate dehydrogenase exhibit 209.2 kJ/M of activation enthalpy and 0.35 kJ/mol K of activation entropy.

We have isolated a water-extracted novel regulator for blood coagulation from an earthworm, Lumbricus rubellus. As a folk remedy, the earthworm has been known to facilitate blood circulation. After complete heat inactivation of endogenous proteases in the earthworm, an anticoagulant(s) was purified through ammonium sulfate fractionation and three consecutive gel permeation chromatography of Sephacryl S-300, Sephadex G-75, and G-150 by measuring activated partial thromboplastin time (APTT) The anticoagulant was further purified to 2,800 fold with a C4 reversed-phase HPLC This activity was stable under heat ($100^{\circ}C$ for 30 min) and acidic conditions (0.4 N HCl). The effects of this partially purified anticoagulant on thrombin were observed with various substrates such as N${\alpha}$-benzoyl-DL-arginine-p-nitroanilide (BApNA), H-D-phenylalanyl-L-pipecoyl-L-arginine-p-nitroanilide (S-2238), N${\alpha}$-p-tosyl-L-arginine methyl ester (TAME), and fibrinogen as a natural substrate. Only TAME hydrolysis, due to an esterase activity of the enzyme, was inhibited among the chromogenic substrates. In addition, the anticoagulant not only inhibited the conversion of fibrinogen to fibrin but also prolonged the fibrin clot formation monitored with the in vitro coagulation test. Based on these observations, we suggest the significance of measuring the ability of antithrombotic drugs to inhibit the esterase activity of thrombin. In this report, it was also shown that the earthworm indeed contained a water-extractable, heat- and acid-stable anticoagulant which could be used as a novel antithrombotic agent.

The NADPH oxidase of phagocytes catalyzes the reduction of oxygen to $O_{2}^{-}$ at the expense of NADPH The enzyme is dormant in resting neutrophils and hecomes activated on stimulation. During activation. $p47^{phox}$ (phagocyte oxidase factor), a cytosolic oxidase subunit, becomes extensively phosphorylated on a number of serines located between S303-S379. Although the biochemical role of phosphorylation is speculative, it has been suggested that phosphorylation could neutralize the strongly cationic C-terminal which may result in the change of conformation of $p47^{phox}$ and subsequent translocation of this protein and other cytosolic components to the membrane. In order to mimic the effect of phosphorylation in terms of neutralizing the positive charges, recombinant $p47^{phox}$ was treated with phenylglyoxal, which removes positive charges of arginine residues. Modification of recombinant $p47^{phox}$ resulted in the activation of oxidase in a cell-free translocation system as well as a conformational change in recombinant $p47^{phox}$ which may be responsible for the activation of the enzyme.

A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiolcontaining oxidation system but not against an oxidation system without thiol. This 25-kDa protein was thus named thiol-dependent protector protein (TPP). The role of TPP in the cellular defense against oxidative stress was investigated in Escherichia coli containing an expression vector with a yeast genomic DNA fragment that encodes TPP (strain YP) and a mutant in which the catalytically essential amino acid in the active site of TPP (Cys-47) has been replaced with alanine by site-directed mutagenesis (strain YPC47A). There was a distinct difference between these two strains in regard to viability, modulation of activities of superoxide dismutase and catalase, and the oxidative damage of DNA upon exposure to menadione. These results suggest that TPP may play a direct role in the cellular defense against oxidative stress by functioning as an antioxidant protein.

Peptidylprolyl cis-trans isomerase (PPlase) catalyzes the cis-trans isomerization of prolyl peptide and facilitates the folding of cellular proteins and peptides. PPlase consists of two distinct immunophilins, each specifically binding to the immunosupressive drug cyclosporin A (CsA) or FK506, respectively. A PPlase was isolated and partially purified from porcine spleen. The molecular weight of porcine spleen PPlase was determined to be ~14,000 on the basis of SDS-PAGE. The purified enzyme was strongly inhibited by FK506, but not by CsA. The inhibition constant and the true concentration of enzyme preparations were determined by active site titration using the tight binding inhibitor FK506: $K_{i}=18.7$ nM and $E_{t}=172$ nM. The equilibrium ratio of conformer. [cis]/[trans], of prolyl peptide substrates (N-Suc-Ala-Xaa-Pro-Phe-p-NA) in anhydrous trifluoroethanol/LiCl solvent system varied from 0.24 to 0.85 depending on the nature of Xaa. Overall. in this solvent-salt system, the populations of the cis conformer of substrates in equilibrium are higher than in an aqueous solution so that the substantial error caused by high background absorption can be reduced. The reactivities of porcine spleen PPlase are shown to be highly sensitive to changes in the structure of substrates. Thus, $k_{cat}/K_m$ value for the most reactive substrate (Xaa Leu) is $4.007+10^{6}M^{1}s^{1}$ and, is 2,636 fold higher than that for the least reactive peptide substrate tested, Xaa=Glu.

Ligand binding properties of muscarinic acetylcholine receptors (mAChRs) in the nematode Caenorhabditis elegans (C. elegans) were characterized by using filtration binding assays. Scatchard analysis using $[^{3}H]N-methylscopolamine$ ($[^{3}H]NMS$) showed that the dissociation constant ($K_d$) and the maximum binding value ($B_{max}$) were $3.3{\pm}0.8{\times}10^{10}$ M and $9.0{\pm}1.1$ fmol/mg protein, respectively. Binding competition experiments indicated that the affinities of C. elegans mAChRs to atropine, scopolamine, and oxotremorine were similar to those of mammalian mAChRs. Pirenzepine binding experiments revealed that the binding pattern of mAChRs in C. elegans closely resembled that of mAChRs in rat brain, suggesting that the receptors consist primarily of Ml subtype. The affinity of mAChRs for oxotrernorine was significantly affected by guanylylimidodiphosphate (Gpp(NH)p), a non hydrolyzable GTP analog, suggesting that mAChRs in C. elegans might be coupled to G proteins. The data presented here indicate the possibility that C. elegans provides a living animal model to study the action mode of the muscarinic cholinergic system.

Astrocyte are the major glial cell type in the central nervous system (CNS), and analogous to macrophage, mediates the number of immune responses such as production of cytokines including tumor necrosis factor alpha ($TNF-{\alpha}$) upon activation. $TNF-{\alpha}$ has been implicated in neuroimmunological disorders through killing oligodendrocytes and thus causing demyelination. It has been previously demonstrated that mitogen-activated T cells synthesized a 26 kDa precursor form of $TNF-{\alpha}$ which is bound to the surface of a membrane, and is later secreted as a 17 kDa mature version. In order to examine whether astrocytes would produce the transmembrane form of $TNF-{\alpha}$, astrocytes were stimulated with biological stimuli and the membrane form of $TNF-{\alpha}$ was analyzed by Western blot and FACS analysis. When astrocytes are stimulated with lipopolysaccharide (LPS), $IFN-{\gamma}/LPS$, or $IFN-{\gamma}/IL-1{\beta}$, they were able to express a membrane-anchored $TNF-{\alpha}$ of approximately 26 kDa protein which was immunoreactive to an $anti-TNF-{\alpha}$ antibody, whereas unstimulated astrocytes or astrocytes treated with $IFN-{\gamma}$ or $IL-1{\beta}$ alone was not. Our FACS data were also consistent with the immunoblot analysis. Our result suggests that the membrane form of $TNF-{\alpha}$ expressed by activated astrocytes may cause local damage to oligodendrocytes by direct cell-cell contact and contribute to demyelination observed in multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE).

The activation of phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine (PC) to phosphatidic acid (PA) and choline in plants as well as animals. To determine the presence of PLD in oat cells, we prepared inside-out plasma membrane and cytosolic fractions from oat tissues. PLD activities in both cytosol and plasma membrane were detected by ion chromatography method. The activity of PLD in plasma membrane was dependent upon $Ca^{2+}$ concentration and was heat stable. To investigate whether G-protein couples to PLD, the effects of $GTP{\gamma}S$ and $GDP{\beta}S$ on the PLD activity were measured. PLD activity was dramatically increased 300~400% in the presence of 50 ${\mu}M$ $GTP{\gamma}S$ but not in the presence of 50 ${\mu}M$ $GDP{\beta}S$. These results indicate that G-protein may be involved in regulation of PLD activity. To identify whether PLD is regulated by red light receptor, phytochrome, we irradiated red, far-red, or red/far-red/red light on oat protoplasts. PLD activity has increased 5-fold and 3-fold by treatment with red light and red/far-red/red light, respectively. In contrast, irradiation with far-red light had little or no effect on PLD activity. These results suggest that phytochrome regulates PLD activity through activation of G-protein in oat cells.

Ferritins from germinated pumpkin seeds were isolated by ammonium sulfate precipitation (0.55 saturation), ion-exchange chromatography on DEAE-cellulose, and gel filtration chromatographies on Sephacryl S-300 and Sephadex G-100. Pumpkin ferritin contains less iron than soybean ferritin. Pumpkin ferritin cross-reacted with anti-soybean ferritin antiserum made in rabbit, and showed two distinct antibody reactive bands, both of equal intensity. The pumpkin ferritins corresponding to the two bands were separable by centrifugation in a sucrose gradient (20~50%). The molecular weights of the native pumpkin ferritins based on the estimation of sucrose gradient centrifugation, gel filtration on Sephacryl S-300 and non-denaturing polyacrylamide gel electrophoresis appeared to be: 530~580 KD (the large molecular weight pumpkin ferritin) and 330-360 KD (the small molecular weight pumpkin ferritin) The large molecular weight pumpkin ferritin contains less iron. Both pumpkin ferritins cross-reacted with anti-soybean ferritin antibody with a spur formation suggesting partial antigenic recognition.

CA(1-8)ME(1-12), the CA-ME hybrid peptide of the amino terminal segments of cecropin A (CA) and melittin (ME), has been reported to have a broad spectrum and improved potency without a hemolytic property. In order to obtain new synthetic peptides with powerful antibacterial activity without hemolytic activity, several hybrid peptides were designed from the sequences of CA, ME, magainin 2, bombinin and lactoferricin. All hybrid peptides were constructed to form an amphipathically basic-flexible-hydrophobic structure and synthesized by the solid phase method. Their hemolytic activities against human red blood cells and antibacterial activities against both Gram-positive and Gram-negative bacteria were detennined. CA(1-8)MA(1-12), CA(1-8)BO(1-12), MA(10-17)ME(1-12) and LF(20-29)ME(1-12) showed comparable activities with broad spectra against both Gram-positive and Gram-negative bacteria relative to CA(1-8)ME(1-12) but without hemolytic properties. These hybrid peptides, therefore, could be useful as model peptides to design a novel peptide with improved antibacterial activity and study on structure-activity relationships of antimicrobial peptides.

Phosphoinositide turnover in response to platelet-derived growth factor, epidermal growth factor, and bradykinin was evaluated in NIH 3T3 cells. Platelet-derived growth factor and bradykinin induced a significant increase in incorporation of $^{32}P$ into phosphatidylinositol (PI), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol 4.5-bisphosphate ($PIP_2$) in serum-starved NIH 3T3 cells. However, epidermal growth factor increased incorporation of $^{32}P$ into these phosphoinositides by only a small amount. Stimulation with platelet-derived growth factor, not bradykinin, caused a rapid elevation of PI and PIP kinase activities that were maximally activated within 10 min. The maximal levels of their elevation in cells with plateletderived growth factor stimulation were 3.2-fold for PI kinase, and 2.1-fold for PIP kinase. Short term pretreatment of NIH 3T3 cells with phorbol 12-myristate 13-acetate, activator of protein kinase C. caused an approximately 60% decrease in platelet-derived growth factor-induced PI kinase activities, indicating the feedback regulation of phosphoinositide turnover by protein kinase C. These results suggest that although the enhancement of phosphoinositide turnover is a rapidly occurring response in platelet-derived growth factor- or bradykinin-stimulated NIH 3T3 cells, phosphoinositide kinases may be associated with initial signal transduction pathway relevant to platelet-derived growth factor but not to bradykinin.

A membrane-bound phosphatidylinositol 4-kinase (PI 4-kinase) was separated in a sucrose gradient and solubilized with 1% Triton X-100 from mouse brain. The enzyme was purified 2,952-fold by various chromatographic techniques including DEAE-cellulose, PI-Sepharose and Sephacryl S-200 gel filtration. The molecular weight of PI 4-kinase was approximately 76 kDa by gel filtration and 70.8 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited specific activity of 11.2 nmol/min/mg protein and pi value of 4.7. Kinetic analysis of the PI 4-kinase indicated apparent $K_m$, values of 190 ${\mu}M$ and 120 ${\mu}M$ for phosphatidylinositol and ATP, respectively. The maximal activity of this purified enzyme was observed at pH 7.4 at an incubation temperature of $37^{\circ}C$. The enzyme activity was significantly activated by $Mg^{2+}$, $Mn^{2+}$ and $Fe^{2+}$, and inhibited severely by $Ca^{2+}$. PI 4-kinase was proved to be pure in its immunoblot test by polyclonal antibody prepared from immunized rabbit sera. By this test, we were able to detect the existence of the same type of PI 4-kinase from other mouse organ tissues, such as liver, heart, kidney and spleen. Furthermore, similar immunoblot analysis with the same antisera recognized the different epitopes of PI 4-kinase proteins from various organs of rabbit, chinese hamster and rat.

Bursts of ethylene production occurred in twice at an early exponential (EEP) and prestationary (PSP) phases, respectively, during growth of callus tissue isolated from the root of soybean seedlings. The second burst of ethylene production at PSP was smaller in magnitude than the earlier one at EEP, but was followed by increases in both guaiacol peroxidase (GuPOX) and ascorbate peroxidase (AsPOX). The increase in AsPOX activity was also preceded by an increase in lipoxygenase (LOX) activity. Treatment of the tissue with the ethylene antagonist 2,5-norbonadiene (NBD) resulted in substantial reduction in LOX and AsPOX activities during this period. GuPOX activity was reduced only slightly, if any, by NBD. Role of ethylene in the sequential induction of LOX and AsPOX in senescing callus tissue is discussed.

Clinical strains of Mycobacterium tuberculosis, M. terrae complex, M. gordonae, M. avium-intracellulae complex, and M. fortuitum from Korean patients were isolated and analyzed by comparing large restriction fragment (LRF) patterns produced by digestion of genomic DNA with infrequent-cutting endonucleases like AsnI and XbaI. and pulsed-field gel electrophoresis (PFGE). Three M. tuberculosis, two M. terrae complex, two M. gordonae, two M. avium-intracellulae complex, and two M. fortuitum strains were compared by using AsnI and XbaI. and this allowed easy visual separation of all epidemiologically unrelated strains. PFGE exhibits different DNA restriction patterns which are easy to compare. Genome size of the strains roughly ranged from 3020 to 3335 kb. The LRF patterns are useful for epidemiologic studies of tuberculosis with regard to drug resistance.