Cleavage of dengue virus NS1-NS2A requires an octapeptide sequence at the C terminus of NS1.

The length of amino acid sequence at the NS1-NS2A juncture of dengue virus that is required for specific cleavage effected by the cis-acting function of NS2A was identified by deletion analysis. Recombinant DNA sequences of NS1-NS2A, each containing a deletion in NS1 followed by a sequence of 3 to 20 amino acids at the C terminus of NS1 preceding the cleavage site, were constructed and expressed with vaccinia virus as a vector. The NS1 product of recombinant vaccinia virus-infected cells was immunoprecipitated and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The occurrence of cleavage between NS1 and NS2A was indicated by the appearance of shortened NS1. Failure to cleave this site yielded a large NS1-NS2A fusion protein. This analysis indicated that a minimum length of eight amino acids at the NS1 C terminus preceding the NS1-NS2A juncture is required for cleavage to take place. Comparison of this eight-amino-acid sequence of the NS1 C terminus of dengue type 4 virus with the analogous sequences of 12 other flaviviruses suggests that the consensus cleavage site sequence is as follows: (table; see text)     

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.     

A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

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