Oil body formation in Euphorbia tirucalli L. cell suspension cultures

The culture of cells of Euphorbia tirucalli L. resulted in the formation of intracellular particles which osmium tetroxide staining and electron microscopy indicated to be oil bodies. Chemical analysis (TLC, HPLC, GLC and GC-MS) disclosed steroids, triterpenoids, and diterpenoids as the major oil components of the cultured cells.     

Pathological evaluation of anti-rheumatic drugs on type II collagen-induced arthritis in DBA/1J mouse

The effects of anti-rheumatic drugs (lobenzarit (CCA); 10 and 50mg/kg, cyclophosphamide (CP); 5 mg/kg and dexamethasone (DM); 0.25mg/kg) were evaluated immunologically and histopathologically on DBA/1J mice that develop polyarthritis after immunization by the intradermal injection of type II collagen. Serum anti-type II collagen IgG levels in the groups treated with CP and DM were significantly suppressed to 1/2 and 1/10 as compared to those of the positive control group, respectively. Those of both groups treated with CCA were not different from those of the positive control group. Histopathological examination revealed that treatment with CP and DM markedly reduced or suppressed inflammatory changes and resulted in low incidence of arthritis. From the standpoint mentioned above, treatment with anti-rheumatic drugs suppressed the development of arthritis in this model, and we could confirm that this model was useful for evaluation of the effect of anti-rheumatic drugs.     

The selective role of cathepsins B and D in the lysosomal degradation of endogenous and exogenous proteins.

A selective inhibitor of cathepsin B, a derivative of E-64 (compound CA-074), and pepstatin-asialofetuin, a potent inhibitor of cathepsin D, were used for an in vivo study of the selective role of these proteinases in lysosomal proteolysis. Administration of compound CA-074 or pepstatinasialofetuin to rats caused only a slight shift of the lysosomal density and no increase in sequestered enzymes in the autolysosomal fraction, although cathepsin B or D activity in the liver was markedly inhibited. These treatments also had little effect on the inhibition of the degradation of endocytosed FITC-labeled asialofetuin. In contrast, leupeptin treatment caused marked inhibition of lysosomal degradation of endogenous and exogenous proteins. These results suggest a small contribution of cathepsins B and D to the initiation of lysosomal proteolysis.     

Heterogeneity of glycoconjugates in the secretory cells of the chinchilla middle ear and eustachian tubal epithelia: a lectin-gold cytochemical study

The present study was conducted to characterize and localize the glycoconjugates in the tubotympanum (auditory or eustachian tube and middle ear cavity) of chinchilla on an ultrastructural level, using lectin-gold complexes with six different lectins: BPA, ConA, RCA-1, WGA, LFA, and SNA. A comparison of the affinity of these lectins demonstrated the heterogeneity of secretory cells. The glandular serous cells and epithelial dark granulated cells produced "serum"-type glycoprotein. The glandular mucous cells and goblet cells produced dominantly "mucin"-type glycoprotein in the light granules, but "serum"-type glycoprotein in the dark cores. The labeling of LFA and SNA showed that sialic acids existed mainly in the mucinous granules of secretory cells and ciliated epithelium glycocalyx, and in the mucous blanket. The results also suggested that the dominant linkage of sialic acids of mucin is a Neu5Ac(alpha 2-6)Gal/GalNAc sequence. Furthermore, the data obtained from ConA and BPA suggested that initial O-glycosylation of mucin took place in the cis side of the Golgi apparatus and that initial N-glycosylation of the serum occurred in the rough endoplasmic reticulum.     

Structure and sequence analysis of the human activin beta A subunit gene.

The cloned genomic DNA containing the human activin beta A subunit gene were analyzed by restriction endonuclease mapping, Southern blotting and DNA sequencing. The activin gene is composed of two exons interrupted by the 9-kb intron. The TATA, CCAAT and CT-stretch sequences were found in the 5'-flanking region of the gene. An intronic sequence contained SV40 enhancer core element in the vicinity of the exon 1. In the 3'-flanking region, we identified eight consensus polyadenylation sequences, five ATTTA motifs, CA element consisting of (CA)14, AP-1 binding site and two SV40 enhancer core elements. A dot matrix analysis revealed the high degree of conservation between the human and rat sequences within the 3'-flanking region, suggesting a possible functional significance.     

Characteristics of mutagenesis by glyoxal in Salmonella typhimurium: contribution of singlet oxygen

The characteristics of mutagenesis by glyoxal in Salmonella tester strains TA100 and TA104, and particularly a possible role of active oxygen species, were investigated. Glyoxal was converted into a non-mutagenic chemical with glutathione (GSH) by glyoxalase I, and the mutagenic activity was enhanced by the depletion of intracellular GSH. Glyoxal caused the reduction of nitro blue tetrazolium, which was suppressed by the addition of 2,5-diphenylfuran, superoxide dismutase (SOD) and catalase (CAT), scavengers of singlet oxygen (1O2), superoxide radical (O2-) and hydrogen peroxide (H2O2), respectively. However, only the 1O2 scavenger almost completely suppressed the mutagenic activity of glyoxal. Mutagenicity assays using strains pretreated with N,N-diethyldithiocarbamate of a SOD inhibitor and strains with low levels of SOD and CAT indicated that the mutagenesis by glyoxal was independent of intracellular levels of SOD and CAT, though glyoxal itself repressed them. Therefore, all the results suggest that 1O2 formed from glyoxal is related to its mutagenesis, but that neither O2- nor H2O2 is intracellularly predominantly related to it. The action of glyoxal against SOD and CAT, and the formation of glyoxal adducts with amino acids as their components are also discussed.     

DNA lesion in rat hepatocytes induced by in vitro and in vivo exposure to glyoxal

The alkaline elution technique was applied to measure the damage of rat hepatic DNA following exposure to glyoxal. DNA single-strand breaks were induced after exposure of primary-cultured hepatocytes to 0.1-0.6 mg/ml glyoxal for 60 min, while no DNA cross-link was observed. Single-strand breaks were also detected in livers of rats within 2 h following a single oral exposure at 200-1000 mg/kg body weight, and the frequency of the breaks reached a maximum around 9 h after exposure. The breaks were almost fully repaired 24 h after exposure to any dose. However, hardly any DNA lesions were detected in other tissues following exposure to 1000 mg/kg glyoxal. Thus, the present results indicate that glyoxal causes DNA single-strand breaks in rat hepatocytes following in vitro and in vivo exposure.     

Late-Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108-15 Cells

The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108-15 cells preincubated with myo-[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15-60 min) of incubation in the cells. PGD2 and PGF2 alpha did not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP) formation at late phase of incubation. The maximum stimulation was obtained at greater than 10(-7) M concentrations of these PGs, the levels being three-and twofold for IP2 and IP1, respectively. 9 alpha, 11 beta-PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2 alpha have no effect up to 10(-6)M. The effects of PGD2 and PGF2 alpha were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation. Inositol 1-monophosphate was mainly identified in the stimulation by 10(-5) M PGD2 and 10(-5) M PGF2 alpha, whereas both inositol 1-monophosphate and inositol 4-monophosphate were produced in the stimulation by 10(5) M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2 alpha and late-phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late-phase effects. These results suggest that PGD2 and PGF2 alpha specifically stimulate hydrolysis of inositol phospholipids.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.