The Association of Ascorbate and Ascorbate Oxidase in the Apoplast with IAA-Enhanced Elongation of Epicotyls from Vigna angularis

The level of (ascorbic acid (AA) plus dehydroascorbic acid (DHA))and the ratio of the level of AA to that of AA plus DHA in intercellularwashing fluid (IWF) of epicotyl segments from Vigna angularisdecreased from 2.8±0.3 to 1.2±0.5nmol (g fr wt)^–1and from 0.23±0.03 to 0.13±0.01, respectively,after incubation of the segments without IAA for 20 h at 27°C.However, these values changed to 5.3±1.7 nmol (g fr wt)^–1and 0.07±0.05 after incubation with 0.1 mM IAA. The activityof cell wall-bound ascorbate oxidase increased by about 20%and 70% after incubation without IAA and with IAA, respectively.However, the activity of cell wall-bound peroxidase was notaffected by incubation with or without IAA. The activities ofascorbate oxidase and peroxidase in IWF decreased by about 85and 75% after incubation without IAA. IAA did not affect thesedecreases to any great extent. A lignin-like compound was formedduring the incubation of epicotyl segments in the absence ofIAA. Formation of this compound was inhibited by IAA. The resultssuggest that one of the causes of the enhancement of elongationgrowth by IAA is the inhibition of peroxidase-dependent lignificationas a result of increases in levels of AA and DHA and in ascorbateoxidase activity. (Received August 16, 1993; Accepted December 6, 1993)     

Reactivities of the coordinated organonitriles in molybdenum(0) and tungsten(0) phosphine complexes: protonation of the nitrile carbon and cleavage of the CN triple bond

The molybdenum and tungsten dinitrogen-organonitrile complexes trans-[M(N₂)(NCR)(dppe)₂] (2, M=Mo; 4, M=W; R=Ph, C₆H₄Me-p, C₆H₄OMe-p, Me; dppe=Ph₂PCH₂CH₂PPh₂) underwent double protonation at the nitrile carbon atom with loss of N₂ and a change in oxidation state to +4 on treatment with hydrochloric acid to afford the cationic imido complexes trans-[MCl(NCH₂R)(dppe)₂]⁺. The solid-state structure of trans-[WCl(NCH₂CH₃)(dppe)₂][PF₆]·CH₂Cl₂ was determined by single-crystal X-ray analysis. Protonation of complexes 2 by fluoroboric acid or hydrobromic acid also formed the similar imido complexes trans-[MoX(NCH₂R)(dppe)₂]⁺ (X=F, Br). In contrast, the dinitrogen complex trans-[Mo(N₂)₂(dppe)₂] reacted with two equiv. of benzoylacetonitrile, a nitrile with acidic CH hydrogen atoms, to give the nitrido complex trans-[Mo(N)(NKCCHCOPh)(dppe)₂] (12), which was accompanied by evolution of dinitrogen and the formation of 1-phenyl-2-propen-1-one in high yields. For complex 12, the zwitterionic structure, where the anionic enolate ligand PhC(O⁺)=CHCN coordinates to the cationic Mo(IV) center through its nitrogen atom, was confirmed by spectroscopic measurements and single-crystal X-ray analysis. A unique intermolecular aromatic C---HO hydrogen bonding was observed in that crystal structure. Complex 12 is considered to be formed via the cleavage of the CN triple bond of benzoylacetonitrile on the metal. A reaction mechanism is proposed, which includes the double protonation of the nitrile carbon atom of the ligating benzoylacetonitrile on a low-valent molybdenum center.     

Distinction between mouse DNA polymerases alpha and beta by tryptic peptide mapping.

Results presented here and in a previous paper (Tanabe et al. (1979) Biochemistry 18, 3401--3406) indicate that mouse beta-polymerase is a single polypeptide with an apparent molecular weight of 40,000. This polypeptide has now been analyzed by tryptic peptide mapping. Comparison of the results with identical analysis of mouse alpha-polymerase reveals that the tryptic peptides derived from the two enzymes are different. These results indicate that beta-polymerase is neither a subunit of alpha-polymerase nor a proteolytic degradation product of alpha-polymerase.     

Polyclonal Antibody Production in Murine Spleen Cells Induced by Staphylococcus

Polyclonal plaque-forming cell (PFC) responses in murine spleen cells induced by Staphylococcus aureus and S. epidermidis were studied. Injection of Balb/c mice with S. aureus strain 248βH resulted in the generation of anti-trinitrophenyl (TNP) and anti-sheep red blood cell PFC in their spleens. Cultures of Balb/c spleen cells in the presence of S. aureus 248βH, Cowan I, or a protein A-deficient mutant yielded many anti-TNP PFC. The larger the number of organisms that were added to the cultures, the better was the PFC response. Both living and killed organisms, were capable of inducing the response, but an excess of living 248βH organisms in the cultures abrogated the response. All of the organisms (12 strains of S. aureus and 11 strains of S. epidermidis) freshly isolated from patients had the ability to induce the polyclonal PFC response in cell cultures. These organisms stimulated cultured C3H/HeJ mouse spleen cells, which were unresponsive to bacterial lipopolysaccharide (LPS). Cultured cells from the spleens of athymic nu/nu mice also responded to these organisms, and the number of PFC in nu/nu cell cultures was always greater than that in nu/+ cells prepared from a haired litter mate. Moreover, the responses of nu/nu spleen cell cultures to which nylon wool column-filtered splenic nu/+ T cells were added were lower than expected. These findings suggest that the polyclonal PFC response to staphylococci is thymus independent, but that the magnitude of the response is regulated by mature T cells. Cultures of macrophage-depleted spleen cells responded to the organisms to an extent similar to that of the control. The 248βH organisms were less capable of stimulating spleen cells of 2-week-old mice (i.e., early maturing B cells) than LPS. However, spleen cells from adult (7-week-old) and aged (9-month-old) mice responded well to both the organisms and LPS. Previous sensitization with the organisms in vivo did not affect any polyclonal responses of spleen cells in vitro to either the organisms or LPS. The role of staphylococcal protein A in the polyclonal PFC response to staphylococci is discussed.     

Inhibiting Effect of Auxin on Glucosamine Incorporation into Cell Walls of Rice Coleoptiles

Auxin induced growth and decreased the hexosamine content ofthe cell walls of rice coleoptile sections. Indole-3-aceticacid (IAA) at 10^–5 M inhibited the incorporation of ¹⁴C-glucosamineinto the cell walls. IAA did not affect the ¹⁴C-incorporationinto the cytoplasm, while inhibitors of glycoprotein synthesis,unicamycin and monensin, suppressed the incorporation into boththe cytoplasm and the cell walls. The radioactivity due to labeledglucosamine in the cell walls increased during the chase, butthis increase was inhibited by IAA. Among the cell wall fractions,the increase in radioactivity and its inhibition by IAA wereconspicuous in the hemicellulose I fraction. The inhibitoryeffect of IAA on glucosamine incorporation into the cell wallswas observed even in the presence of 0.15 M mannitol solutionwhich completely suppressed the IAA-induced growth. These resultssuggest that auxin induces growth at least partly by inhibitingthe transport of asparagine-linked glycoproteins from the cytoplasmto the cell walls. ¹ Present address: Department of Biology, Faculty of Science,Osaka City University, Sumiyoshi-ku, Osaka 558, Japan (Received July 23, 1986; Accepted December 22, 1986)     

Allelic dimorphism in a surface antigen gene of the malaria parasite Plasmodium falciparum

Merozoites of the malaria parasite Plasmodium falciparum carry surface proteins processed from a precursor termed p190 or p195. Polymorphism has been reported in this protein. Since the protein is a candidate for a malaria vaccine, it is important to understand the nature of this polymorphism. We have determined the complete nucleotide sequence of the p190 gene from the MAD20 strain (a Papua New Guinea isolate). Comparisons of the gene with that from other strains of P. falciparum allowed us to study the genetic basis of the antigen's polymorphism. The gene consists of sequences distributed in variable blocks, which are separated by conserved or semi-conserved sequences. Variable sequences occur both in regions that code for tripeptide repeats and in regions with no apparent repeats. Interestingly, according to the present data, variable sequences are not widely polymorphic but fall into two distinct types. We argue that the p190 protein is encoded by dimorphic alleles capable of limited genetic exchange and present evidence at the nucleotide level documenting intragenic recombination in Plasmodium.