Purification and characterization of multicatalytic proteinase from eggs of the ascidian Halocynthia roretzi

A multicatalytic (high-molecular-weight) proteinase has been purified from eggs of the ascidian Halocynthia roretzi by a procedure including column chromatographies on DEAE-cellulose and hydroxylapatite and gel filtration on Sepharose 6B. The purified enzyme seemed to be homogeneous, as judged by disc-polyacrylamide gel electrophoresis, isoelectrofocusing, sedimentation velocity, and gel filtration. The molecular weight of the enzyme was estimated to be 610,000 by gel filtration. The isoelectric point and the sedimentation coefficient (S20,w) were 6.2 and 22.8S, respectively. The enzyme showed several protein bands with molecular weight ranging from 25,000 to 33,000 on SDS-polyacrylamide gel electrophoresis and a cylindrical or ring-like structure composed of several subunits under the electron microscope, indicating that the enzyme exists as a large molecule consisting of several protein components. The enzyme exhibited chymotrypsin-like and trypsin-like activities whose pH optima were both 7.0. Chymostatin and its analog, calpain inhibitor I, and elastatinal inhibited both activities, whereas leupeptin and antipain only inhibited the latter. The former activity was stimulated by a low concentration of SDS or fatty acid, whereas the latter was not. Thus, the properties of the enzyme purified from ascidian eggs are similar to those of multicatalytic proteinases from mammalian tissues.     

A large impact of tropical biomass burning on CO and CO2 in the upper troposphere

A large interannual variation of biomass burning emissions from Southeast Asia is asso-ciated with the ENSO events. During 1997/98 and 1994 El Nino years, uncontrolled wildfires of tropical rainforests and peat lands in Indonesia were enlarged due to a long drought. EnhancedCO injection into the upper troposphere from the intense Indonesian fires was clearly observed in the 8-year measurements from a regular flask sampling over the western Pacific using a JAL air-liner between Australia and Japan. This airliner observation also revealed that upper tropospheric CO_2 cycle largely changed during the 1997 El Nino year due partly to the biomass burning emis-sions. Widespread pollution from the biomass burnings in Southeast Asia was simulated using aCO tracer driven by a 3D global chemical transport model. This simulation indicates that tropical deep convections connected to rapid advection by the subtropical jet play a significant role in dis-persing biomass-burning emissions from Southeast Asia on a global scale.     

Complete nucleotide sequence of the prophage VT2-Sakai carrying the verotoxin 2 genes of the enterohemorrhagic Escherichia coli O157:H7 derived from the Sakai outbreak

The enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain RIMD 0509952, derived from an outbreak in Sakai city, Japan, in 1996, produces two kinds of verotoxins, VT1 and VT2, encoded by the stx1 and stx2 genes. In the EHEC strains, as well as in other VT-producing E. coli strains, the toxins are encoded by lysogenic bacteriophages. The EHEC O157:H7 strain RIMD 0509952 did not produce plaque-forming phage particles upon inducing treatments. We have determined the complete nucleotide sequence of a prophage, VT2-Sakai, carrying the stx2A and stx2B genes on the chromosome, and presumed the putative functions of the encoded proteins and the cis-acting DNA elements based on sequence homology data. To our surprise, the sequences in the regions of VT2-Sakai corresponding to the early gene regulators and replication proteins, and the DNA sequences recognized by the regulators share very limited homology to those of the VT2-encoding 933W phage carried by the EHEC O157:H7 strain EDL933 reported by Plunkett et al. (J. Bacteriol., p1767-1778, 181, 1999), although the sequences corresponding to the structural components are almost identical. These data suggest that these two phages were derived from a common ancestral phage and that either or both of them underwent multiple genetic rearrangements. An IS629 insertion was found downstream of the stx2B gene and upstream of the lysis gene S, and this might be responsible for the absence of plaque-forming activity in the lysate obtained after inducing treatments.     

Prostaglandin E(2) production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E(2) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE(2) production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC(50) of 1.0 microM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE(2) production by activated macrophages.     

Synthesis of epidermal growth factor (EGF) receptor in vitro using SP6 RNA polymerase-transcribed template mRNA

The epidermal growth factor (EGF) receptor plays a key role in the control cellular proliferation, and its homology to the avian erythroblastosis virus erb B oncogene implicates its involvement in cellular transformation. The establishment of a correlation between the various structural domains of the EGF receptor and their functional counterparts would greatly advance our understanding of these processes. To this end, we have constructed an expression vector containing the SP6 viral promoter and an adjacent cDNA fragment encoding the full-length EGF receptor. Upon addition of SP6 RNA polymerase, this DNA is capable of generating large amounts of EGF receptor mRNA; this RNA can then be translated in vitro into immunoprecipitable EGF receptor protein. The translational efficiency of this EGF receptor RNA was found to be relatively low: approx. 100-fold lower than globin RNA synthesized using SP6 RNA polymerase. Use of these tools should now permit the synthesis and analysis of mutated EGF receptor protein in an effort to clarify the role of this receptor in growth control.     

Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

Dioxin silences gonadotropin expression in perinatal pups by inducing histone deacetylases: a new insight into the mechanism for the imprinting of sexual immaturity by dioxin

Maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes the impairment of reproduction and development in the pups. Our previous studies have revealed that maternal treatment with TCDD attenuates the fetal production of pituitary gonadotropins (luteinizing hormone (LH) and follicle-stimulating hormone) at gestational day (GD) 20, leading to the impairment of sexual behavior in adulthood. However, the mechanism underlying such a reduction has remained unknown until now. When pregnant rats at GD15 were given an oral dose of TCDD (1 μg/kg), the testicular expression of steroidogenic proteins was reduced between GD20 and postnatal days (PND) 2. In accordance with this, the pituitary expression of gonadotropin β-subunit and serum gonadotropin were also attenuated from GD20 to PND0 in a pup-specific fashion. To identify the target genes linked to a fetal reduction in gonadotropin β-subunit, we performed a DNA microarray analysis using the fetal pituitary and its regulatory organ, the hypothalamus. The results obtained showed that TCDD induced histone deacetylases (HDACs) in the fetal pituitary. In support with this, TCDD markedly deacetylated histones H3 and H4 twined around the promoter of the fetal LHβ gene. This effect was fetus- and LHβ-specific, and this was not observed in the maternal pituitary or for other pituitary hormone genes. Finally, an LHβ reduction caused by TCDD was completely restored by maternal co-treatment with valproic acid, an HDAC inhibitor. These results strongly suggest that the increased deacetylation of histone owing to HDAC induction plays a critical role in the TCDD-induced reduction in LHβ in the fetal pituitary.     

Characterization of acrolein-induced protein cross-links

Lipid peroxidation products contribute to protein aggregation that occurs during oxidative stress in a number of degenerative disorders. Acrolein (ACR), a highly toxic lipid peroxidation aldehyde, is a strong cross-linking agent of cellular components such as proteins. To understand the mechanisms of oxidative stress-induced protein aggregation, this study characterized the ACR modification of chain B from bovine insulin by mass spectrometry. To identify the cross-linking sites, the ACR-treated peptide was digested with a protease and the resulting peptides were analysed by liquid chromatography-tandem mass spectrometry. Inter- and intra-molecular cross-linking adducts were identified between amino groups and the side chain of histidine in the peptide. These results indicated that the ACR-induced cross-links were accompanied by two reactions, namely Michael addition and Schiff base formation. In conclusion, the use of mass spectrometric techniques provided chemical evidence for protein cross-linking with ACR.     

Inhibition of topoisomerase I by heparin

DNA topoisomerase I isolated from mouse mammary cacinoma cells was shown to be inhibited by heparin, the dose giving 50% inhibition (IC₅₀) being 0.20 μg/ml. Other chemically related acid mucopolysaccharides including heparan sulfate, dermatan sulfate etc. were more than 500 times less active than heparin. When the amount of enzyme was doubled relative to the substrate the inhibition was reversed. Addition of heparin to assay mixtures after the initiation of the reaction immediately inhibited the enzyme reaction.