Ontogenesis of α2-Adrenoceptor Coupling with GTP-Binding Proteins in the Rat Telencephalon

The ontogenesis of alpha 2-adrenoceptors and GTP-binding proteins and their coupling activity were investigated in telencephalon membranes of developing rats. The manganese-induced elevation of [3H]clonidine binding was increased in an age-dependent manner but the guanosine 5'-O-(3-thio)triphosphate-induced decrease in binding did not change. The extent of the binding of [3H]clonidine at 15 nM (saturable concentration) increased in an age-dependent manner and reached the adult level at 4 days after birth. Cholera toxin and pertussis toxin catalyzed ADP-ribosylation of proteins of 46 and 41/39 kilodaltons (kDa) in solubilized cholate extracts of the membranes. The 41/39-kDa proteins ADP-ribosylated by pertussis toxin (Gi alpha + Go alpha) were increased with age and reached the adult level at day 12, whereas the 46-kDa protein (Gs alpha) reached its peak on day 12 and then decreased to the fetal level at the adult stage. The immunoblot experiments of the homogenates with antiserum (specific antibody against alpha- and beta-subunit of GTP-binding proteins) demonstrated that the 39-kDa alpha-subunit of (Go alpha) and the 36-kDa beta-subunit of GTP-binding protein (beta 36) increased with postnatal age. In contrast, 35-kDa beta-subunit (beta 35) did not change. From these results, it is suggested that the coupling activity of alpha 2-adrenoceptor with GTP-binding protein gradually develops in a manner parallel with the increase of alpha 2-adrenoceptor and pertussis toxin sensitive GTP-binding proteins, Gi, and that alpha 39 beta 36 gamma may be related to the differentiation and/or growth of nerve cells in rat telencephalon.     

Close linkage of MEN2A with RBP3 locus in Japanese kindreds

Summary The gene responsible for multiple endocrine neoplasia type 2A (MEN2A) has recently been assigned to the pericentromeric region of chromsome 10 in European Caucasian kindreds by linkage analysis using a DNA marker, interstitial retinol-binding protein 3 (RBP3). We have found tight linkage between the MEN2A and RBP3 loci in Japanese MEN2A kindreds. The maximum lod score is 5.19 at a recombination fraction of 0.00. This result suggests that mutation of a certain gene close to RBP3 is responsible for MEN2A irrespective of ethnic backgrounds.     

Antimetastatic effect of endogenous tumor necrosis factor induced by the treatment of recombinant interferon γ followed by an analogue (GLA-60) to synthetic lipid A subunit

Summary We have investigated the effect of endogenous production of tumor necrosis factor (TNF) induced by the combination of recombinant interferon (rIFN) as a primer followed by GLA-60 as a trigger (rIFN/GLA-60) on murine lung metastases caused by B16-BL6 melanoma. In order to examine the therapeutic effect of endogenous TNF on tumor metastasis, the ability of multiple administrations of rIFN/GLA-60 to induce TNF production was also tested. The multiple administrations of rIFN/GLA-60 at intervals of 2 days were effective for the induction of endogenous TNF in mice but continuous multiple administrations of them for 2–4 days were not. In tumor-bearing mice, the production of endogenous TNF by rIFN/GLA-60 was less than that of normal mice, but treatment 3 days after the surgical excision of primary tumors showed the endogenous TNF production to be similar to that in normal mice. In the experimental lung metastasis model, intravenous administration of rIFN followed by intravenous or intranasal administration of GLA-60 showed potent inhibition of lung metastases of B16-BL6 melanoma, whereas the reverse sequence of administration (GLA-60/rIFN) or administration of a mixture of rIFN and GLA-60, which cannot induce the production of TNF, caused no inhibition of lung metastases. These results indicated that the regression of tumor metastases by rIFN/GLA-60 was mediated by the production of endogenous TNF in addition to the direct effects of both immunostimulants. Furthermore, the administration of rIFN and GLA-60 significantly inhibited the tumor metastases in spontaneous lung metastasis model. These results may provide a promising approach for the treatment of cancer metastasis as a result of its ability to induce endogenous TNF.     

Combined action of paraquat and superoxide on the peroxidation of detergent-dispersed linolenic acid.

We investigated the peroxidative effect of paraquat and active oxygens on detergent-dispersed linolenic acid in phosphate buffer (pH 7.5) from the malondialdehyde (MDA) level. Our complete system and further inclusion of catalase were effective in stimulating MDA formation. On the other hand, xanthine oxidase (XOD) or paraquat omission, superoxide dismutase (SOD) inclusion or anaerobic incubation inhibited the formation of MDA. Ferrous ion was weakly associated with phosphate of the buffer, forming a complex, and the release of ferrous ion from the complex intensified the MDA levels with the complete and catalase inclusion systems. The electron paramagnetic resonance (EPR) spectra using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) showed that superoxide, produced immediately after the addition of XOD, played a crucial role. We could obtain a DMPO-OOH signal at the starting stage whenever MDA stimulation was observed. The omission of paraquat, however, produced no increase in MDA level in spite of an appearance of DMPO-OOH signal, indicating that paraquat also plays an important role. On the other hand, Desferal, a ferric chelator, showed a concentration-dependent inhibition effect. There was an immediate strong intensity of DMPO-OOH and paraquat signals. We did not, however, observe MDA stimulation at 250 microM Desferal, which confirms that ferrous ion plays an essential role in the lipid peroxidation. These results indicate a combined action of paraquat (or its radical) and superoxide on the accessibility of ferrous ion, including its release from the complex with phosphate, which may be an endogenous chelator. The possibility of ternary complex participation is also discussed.     

Transformation ofBacillus subtilis with the treatment by alkali cations

Summary ExposingBacillus subtilis cultures to high concentrations of alkali cations, especially K⁺, allows efficient transformation by plasmids. The method allows transformation with unfractionated plasmid DNA, monomeric plasmid DNA as well as linear plasmid DNA.B. subtilis strains, not amenable to natural transformation, were also transformed by the present method.     

Restricted movement of the fluvial form of red-spotted masu salmon,Oncorhynchus masou rhodurus,in a mountain stream,Central Japan

Movement of the fluvial form of red-spotted masu salmon (1⁺ and older),Oncorhynchus masou rhodurus, was studied using mark-recapture methods in a Japanese mountain stream. Most (63–91%) adult salmon were recaptured in the pool in which they were marked. The rest of the salmon moved upstream or downstream <20m during the non-breeding period. The proportion of the salmon moving increased slightly during the breeding period, but did not exceed 66%. The distance moved was also more variable during this period. The proportion of the smaller salmon which moved was larger than that of the larger fish during the non-breeding period. Conversely, during the breeding period, larger fish moved more frequently. Sedentary behaviour and local movements of adult salmon seem to be affected by their social relationships.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139     

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A¹ (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.     

Highly repetitive sequences and characteristics of genomic DNA in unicellular cyanobacterial strains

Abstract Microcystis aeruginosa (Synechocystis ) is a unicellular cyanobacterium that performs oxygenic photosynthesis. We found two novel sets of repetitive sequences, A (REP-A) and B (REP-B), on the M. aeruginosa K-81 genomic DNA, which consisted of distinct motifs of tandem repeated sequences located in the up- and downstream regions of the orf1 structural gene, respectively. Genomic Southern hybridization revealed multicopies of REP-A and -B on the genome. Furthermore, genomic Southern blots of cyanobacteria species with the REP-A and -B probes revealed that different hybridization signals appeared on the genomic DNAs of all 12 Microcystis strains, but no signal appeared on those of Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, and Anabaena sp. PCC 7120.