Incorporation of Serine-3-14C into Sphingolipid by Rat Liver Particulate

Four semi-synthetic and fourteen quassinoids were tested for their antifeedant and insecticidal activity against 3rd instar larvae of the diamondback moth (Plutella xylostella). In this quassinoid series, isobrucein-B was the most potent compound in both assays. Chemical conversion of the methoxy and/or methylenedioxy groups in the A and C rings to hydroxy groups among these quassinoids resulted in decreased activity.     

Studies on the Structure of Gluco-oligosaccharides Obtained from the Partial Acid Hydrolyzate of Sclerotia of Sclerotinia libertiana

The defatted sclerotia powder was partially hydrolyzed with dilute acid, and the material obtained was fractionated by carbon column chromatography, separated into two disaccharides, three trisaccharides and three tetrasaccharides, respectively. In these hydrolyzates α, α-trehalose, Iaminaribiose, and gentiobiose were identified.     

Requirement of caspase and p38MAPK activation in zinc-induced apoptosis in human leukemia HL-60 cells.

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL-60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL-60 cells with Zn for 6 h in the presence of Py (1 micro m) exhibited cytotoxicity in a Zn dose-dependent manner (25-200 micro m). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose-dependent fashion (50-100 micro m), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method, peaked at 25 micro m, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a cell-permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 micro m Py and 25 micro m Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP-ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen-activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z-Asp-CH2-DCB, a broad-spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK-specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z-Asp-CH2-DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.     

Modification of regression of virally xenogenized tumor cells by cyclophosphamide and busulfan

Summary Rat fibrosarcoma cells infected with Friend leukemia virus (FV-KMT-17) grow for a short time and then regress spontaneously in syngeneic hosts. This regression mechanism was examined by analyzing the immunomodulating action of the antitumor drugs busulfan (BU) and cyclophosphamide (CY). In preliminary experiments, the optimum dosages of BU and CY for the enhancement of DTH responses to SRBC were 10 mg/kg and 40 mg/kg respectively. Treatment of rats with BU (10 mg/kg) on day 5 induced the regression of KMT-17 cells, while in contrast, the same drug delayed the spontaneous regression of FV-KMT-17 cells. Pretreatment with CY (40 mg/kg) on day 5 did not affect the growth of KMT-17 or FV-KMT-17 cells. After the same treatment schedule, BU inhibited humoral antibody formation against SRBC and against virus-associated antigen (VAA), NK cell activity, and ADCC effector cell activity. On the other hand, CY did not affect the activities of NK cells or ADCC effector cells, although it significantly augmented the DTH responses to SRBC and the production of antibody to VAA but had no effect on production of antibodies to SRBC. These results suggest that NK cells and ADCC may play an important role in the initial stage of the spontaneous regression of FV-KMT-17 cells.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education Abbreviations used: BU, busulfan; CY, cyclophosphamide; PFC assay, plaque forming cell assay; VAA, virus-associated antigen; NK cell, natural killer cell; ADCC, antibody dependent cellular cytotoxicity; MuLV, murine leukemia virus; DTH, delayed type hypersensitivity; SRBC, sheep red blood cells; C.I., cytotoxic index; CRBC, chicken red blood cells; IL-1, interleukin 1; IL-2, interleukin 2; IFN, interferon     

Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.     

Freeze denaturation of enzymes and its prevention with additives

Freeze inactivation of LDH, MDH, ADH, G-6-PDH, and PK and its prevention with additives such as sodium glutamate and albumin were studied. LDH, MDH, ADH, G-6-PDH, and PK, each lost their activity during frozen storage at -20 degrees C. The speed of the inactivation differed in each. The stability of the enzymes increased with the increase of the enzyme concentration. Sodium glutamate and albumin prevented the freeze inactivation. While the activity of the LDH solution frozen without additives was almost lost during a day of frozen storage, those frozen with either glutamate (0.2 M) or albumin (0.1%) added decreased less quickly. The residual activity after 1 day was 50% the initial prefreeze value for the former and 10% for the latter, respectively. Combined use of glutamate and albumin prevented the inactivation the best and maintained the initial activity almost completely over 6 weeks. The enzymes tested lost some part of their activity when their solutions were diluted by the media. This inactivation was prevented to a significant extent by the addition of sodium glutamate and/or albumin to the diluting media.     

Enhanced susceptibility of target KMT-17 cells to activated macrophages by treatment with the antitumor agent bleomycin

Summary We observed that after KMT-17 cells had been treated with bleomycin (BLM), even with a dose as high as 160 g/ml, they were still able to form colonies in soft agar. We then studied the susceptibility of KMT-17 cells treated with BLM to activated macrophages. During a colony inhibition assay, BLM-treated KMT-17 cells were found to be much more susceptile to activated macrophages than nontreated KMT-17 cells, moreover, a tumor neutralizing assay showed that the growth of BLM-treated KMT-17 cells was also significantly inhibited by activated macrophages as compared with nontreated KMT-17 cells. Macrophages activated by both BLM and the Nocardia rubra cell wall skeleton were able to mediate such tumor inhibition activity in BLM-treated KMT-17 cells. Activated macrophages did not seem to have strong antitumor activity against nontreated KMT-17 cells in vivo, however, the life span of the rats which were inoculated i. p. with KMT-17 cells was significantly expanded after the tumorbearing rats were given BLM i.p. The data presented here suggest that not only does BLM have a direct tumoricidal effect on KMT-17 cells, it also regulates immunosensitivity of targets to immune effectors. We also discuss the mechanism for enhancing the susceptibility of KMT-17 cells to activated macrophages brought about by treatment with BLM.Supported in part by a Grant-in-Aid for Cancer Research from the Japanese Ministry of Education, Science, and Culture     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia. 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.     

The role of polypyrrole as charge transfer mediator and immobilization matrix for d-fructose dehydrogenase in a fructose sensor

A fructose dehydrogenase (FDH) modified electrode is produced by the electroadsorption of a layer of FDH on a platinum electrode followed by the electropolymerization of a polypyrrole (PPy) film around and over the enzyme. This immobilizes and stabilizes the enzyme as well as providing an electron transfer pathway to the electrode. The amperometric response to fructose and the enzymatic activity are measured as a function of PPy film thickness. The electrode is shown to have a maximum response at a PPy thickness of approximately the thickness of the enzyme layer. A measure of the electrode efficiency is also obtained, this is the amperometric response to fructose as a percentage of that expected on the basis of the enzyme activity. The functioning of the electrode is also dependent on the counter-ion used for PPy polymerization. This is shown to be mainly related to the nucleation and growth of the PPy film in the interfacial region.