Isolation and characterization of a protease inhibitor from spinach leaves

An acid-stable and heat-labile proteinous protease inhibitor which was found in spinach leaves but not in seeds was isolated by sequential chromatography and preparative isoelectric focusing. The isoelectric point of this inhibitor was 4.5. The inhibitor had a M_r of ca 18 000 and was rich in aspartic acid and glycine; it had 4 half-cystine, 2 tryptophan and no methionine residues. Its extinction coefficient (E|_cm^%) was 13.7 at 280 nm. The inhibition was competitive and the dissociation constant was 3.32 × 10^?13 M. The inhibitor was specific to serine proteases and strongly inhibited trypsin and weakly inhibited α-chymotrypsin and kallikrein.     

Novel DNA photocleaving agents with high DNA sequence specificity related to the antibiotic bleomycin A2.

We have designed and synthesized a series of novel DNA photocleaving agents which break DNA with high sequence specificity. These compounds contain the non-diffusible photoactive p-nitrobenzoyl group covalently linked via a dimethylene (or tetramethylene) spacer to thiazole analogues of the DNA binding portion of the antibiotic bleomycin A2. By using a variety of 5' or 3' 32P-end labeled restriction fragments from plasmid pBR322 as substrate, we have shown that photoactive bithiazole compounds bind DNA at the consensus sequence 5'-AAAT-3' and induce DNA cleavage 3' of the site. Analysis of cleavage sites on the complementary DNA strand and inhibition of DNA breakage by distamycin A indicates these bithiazole derivatives bind and attack the minor groove of DNA. A photoactive unithiazole compound was less specific inducing DNA breakage at the degenerate site 5'-(A/T)(AA/TT)TPu(A/T)-3'. DNA sequence recognition of these derivatives appears to be determined by the thiazole moiety rather than the p-nitrobenzoyl group: use of a tetramethylene group in place of a dimethylene spacer shifted the position of DNA breakage by one base pair. Moreover, much less specific DNA photocleavage was observed for a compound in which p-nitrobenzoyl was linked to the intercalator acridine via a sequence-neutral hexamethylene spacer. The 5'-AAAT-3' specificity of photoactive bithiazole derivatives contrasts with that of bleomycin A2 which cleaves DNA most frequently at 5'-GPy-3' sequences. These results suggest that the cleavage specificity exhibited by bleomycin is not simply determined by its bithiazole/sulphonium terminus, and the contributions from other features, e.g. its metal-chelating domain, must be considered. The novel thiazole-based DNA cleavage agents described here should prove useful as reagents for probing DNA structure and for elucidating the molecular basis of DNA recognition by bleomycin and other ligands.     

Cloning and sequencing of the genes for A and B subunits of the V-type Na(+)-ATPase of a facultatively anaerobic alkaliphile

The structural genes for A and B subunits of the V-type Na(+)-ATPase from a facultatively anaerobic alkaliphile (Amphibacillus sp.), strain M-12, were cloned and sequenced. Transformation of Escherichia coli with the genes overexpressed two proteins, which crossreacted with an antiserum against A and B subunits of the V-type Na(+)-ATPase from Enterococcus hirae. The deduced amino acid sequence (594 amino acids; Mr, 66,144) of A subunit of the M-12 enzyme exhibited 73%, 51%, 49% and 53% identities with those of V-type ATPases from E. hirae, Thermus thermophilus, Neurospora crassa and Drosophila melanogaster, respectively. The amino acid sequence (458 amino acids; Mr, 51,308) of B subunit of the M-12 enzyme was 74%, 53%, 52% and 54% identical with those of the ATPases from E. hirae, T. thermophilus, N. crassa and D. melanogaster, respectively. The fact indicates that the amino acid sequences of A and B subunits of the M-12 enzyme exhibit significantly higher homologies with those of the E. hirae Na(+)-ATPase as compared with those of the H(+)-ATPases from T. thermophilus, N. crassa and D. melanogaster.     

Tobamovirus-resistant tobacco generated by RNA interference directed against host genes

Two homologous Nicotiana tabacum genes NtTOM1 and NtTOM3 have been identified. These genes encode polypeptides with amino acid sequence similarity to Arabidopsis thaliana TOM1 and TOM3, which function in parallel to support tobamovirus multiplication. Simultaneous RNA interference against NtTOM1 and NtTOM3 in N. tabacum resulted in nearly complete inhibition of the multiplication of Tomato mosaic virus and other tobamoviruses, but did not affect plant growth or the ability of Cucumber mosaic virus to multiply. As TOM1 and TOM3 homologues are present in a variety of plant species, their inhibition via RNA interference should constitute a useful method for generating tobamovirus-resistant plants.     

Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: a new strategy for generating completely non-fucosylated recombinant therapeutics

Currently, removal of core fucose from the Fc oligosaccharides of therapeutic antibodies is widely recognized as being of great importance for the effector function of antibody-dependent cellular cytotoxicity, and alpha-1,6-fucosyltransferase (FUT8) knockout cells have been generated as an ideal host cell line for manufacturing such therapeutics. Here, we attempted to identify genes other than FUT8 that could be targeted for the manufacture of non-fucosylated therapeutics. Loss-of-function analyses using siRNAs against three key genes involved in oligosaccharide fucosylation in Chinese hamster ovary (CHO) cells revealed that there was a positive correlation between the Fc oligosaccharide fucosylation and the mRNA expression through the origin in the cases of both GDP-fucose 4,6-dehydratase (GMD) and FUT8, but not for the GDP-fucose transporter, suggesting that there is no functional redundancy in GMD and FUT8. GMD knockout CHO/DG44 cells were successfully established, and were confirmed to be devoid of intracellular GDP-fucose and to produce completely non-fucosylated antibodies. GMD knockout cells recovered their fucosylation capability through the salvage pathway upon addition of l-fucose into the culture medium, and exhibited equable morphology, growth kinetics and recombinant protein productivity, demonstrating that loss of oligosaccharide fucosylation has no impact on these cellular phenotypes. Our results demonstrate that GMD knockout is a new strategy applicable to the manufacture of non-fucosylated therapeutic antibodies, and completely O-fucose-negative therapeutics as well.     

Phenotypic diversity of 188 rice embryo mutants

We have identified 188 embryo mutants of rice and characterized them into six groups based on their phenotypes: (1) embryoless in mature seed, (2) deletion of embryonic organ(s), (3) abnormal position of embryonic organs, (4) abnormal embryo size, (5) defect in organ morphology, and (6) variable abnormal phenotypes in spite of single mutations. Three types of organless mutants are obtained: small globular embryo, club-shaped embryo, and large embryo. Although 12 shootless mutants derived from at least three loci are identified, only three radicleless mutants are recovered, which produce normal adventitious roots after germination. In reduced embryo mutants, every embryonic organ is reduced, in contrast to giant embryo mutants in which only scutellum is enlarged. Considerable number of mutants are categorized into (5) and (6) in the above. These diverse embryo mutants would serve as promising materials for genetic study of embryogenesis. © 1995 Wiley-Liss, Inc.     

Mechanism of haemolysis by Vibrio vulnificus haemolysin

The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.     

SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells that are regulated by the Wnt signaling pathway

Identification and expansion of retinal progenitor cells are critical issues from both scientific and clinical aspects. Here, we identified SSEA-1 (CD15) as a novel surface antigen that can be used to define immature retinal progenitor cells. SSEA-1-expressing retinal cells were found in the peripheral region of the early embryonic mouse retina, and then their number dramatically disappeared along with retinal development. FACS analysis showed that the cells strongly positive for SSEA-1 co-expressed Ki67 proliferation antigen in all the developmental stages examined. The SSEA-1-expressing cells formed larger colonies than the non-expressing ones in retinal re-aggregation cultures. Moreover, late onset of rhodopsin expression was observed in SSEA-1-positive progenitor cells, supporting the idea that these cells have an intrinsically immature character. Differential expression of Wnt signal-related genes between SSEA-1-positive and -negative subpopulations of retina cells was revealed, and the expression of constitutively active forms of Wnt signaling molecules resulted in a greater number of SSEA-1-positive cells. In light of all of the data taken together, we propose SSEA-1 to be a surface marker to define a regionally restricted immature subset of progenitor cells of mouse neural retina, with SSEA-1 expression by them positively regulated by Wnt signals.     

The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.