In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

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.     

Ibaraki Virus,an Agent of Epizootic Disease of Cattle Resembling Bluetongue

Replication of Ibaraki virus was not inhibited by 5-iodo-2′-deoxyuridine, indicating that the virus is an RNA virus. The virus was resistant to ether, chloroform and deoxycholate, sensitive to trypsin, very labile at acidic pH but stable at pH 6.4 or higher, and was resistant to repeated freezing and thawing. The virus was readily inactivated at 56 C or higher, was fairly stable at 37 C, and very stable at 4 C, while it rapidly lost infectivity when stored frozen at —20 C. The virus was readily sedimented by centrifugation at 40 000Xg for 60 min. It readily passed through membrane filters of 200 mμ pore size, passed through 100 μfilters but only with some titer loss and did not through 50 mμ filters. In these tests, the bluetongue virus used as a control behaved in the same manner as Ibaraki virus. These findings provide additional evidence for the similarity of Ibaraki virus to bluetongue virus which had been previously demonstrated on the basis of seasonal incidence, symptomatology and pathology of the diseases caused by these viruses and the behavior of the viruses in cell cultures, embryonated eggs and laboratory animals. The present study, however, provided no evidence for any serological relation between these two viruses. More Information is needed to reach a final decision on the classification of Ibaraki virus, particularly regarding the morphology of the virion, the doublestrandedness of the viral RNA and other basic features.     

Evidence for acrosin-like enzyme in sperm extract and its involvement in fertilization of the ascidian,halocynthia roretzi

The presence of a protease has been demonstrated in sperm of the solitary ascidian, Halocynthia roretzi, by using t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) and other arginyl or lysyl MCA derivatives as substrates. Several properties of the enzyme were investigated in a crude extract. The activity had a pH optimum near 8.0 and was enhanced by the addition of CaCl₂. The Km value of 87μM was determined for Boc-Val-Pro-Arg-MCA under the optimal conditions. An apparent molecular weight was estimated to be 35,000 by gel filtration. The enzyme was inhibited with diisopropyl fluorophosphate, leupeptin, antipain, p-aminobenzamidine, Val-Pro-Arg-CH₂Cl, and soybean trypsin inhibitor, but scarcely inhibited with chymostatin, elastatinal, p-chloromercuribenzoic acid, tosyl-Lys-CH₂Cl, and tosyl-Phe-CH₂Cl. Boc-Val-Pro-Arg-MCA, the most susceptible of the substrates examined, showed the most effective inhibition against fertilization of ascidian eggs. Thus, this enzyme in ascidian sperm extract has features closely similar to mammalian acrosin [EC 3.4.21.10], and we conclude that the enzyme is involved in fertilization as one of the lysins.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

Effects of fat content in the diet on hepatic peroxisomes of the rat

Effects of fat content in the diet on rat liver peroxisomes was examined. In the livers of rats fed for one week on the high-fat diet containing 30% fat, the cyanide-insensitive palmitoyl-CoA oxidation was accelerated to eight times that of control and the enzymic activities of catalase, carnitine acetyltransferase and carnitine palmitoyltransferase were elevated by the factors of 1.3, 5 and 2, respectively. In contrast, the activities of D-amino acid oxidase in addition to the three enzymes mentioned above were all lowered by 20% when the animals were maintained on a fat-free diet for the same period of time. It appears that the high-fat diet-induced increase in the activity of carnitine palmitoyltransferase is a result of the raised activity of this enzyme in mitochondria only while the apparent high activity reflects stimulation of carnitine acetyltransferase in all the subcellular fractions. Another notable effect of the high-fat diet was a remarkable increase in the quantity of a peroxisome-associated polypeptide which was separable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is noteworthy that this effect of the high-fat diet resemble that of clofibrate. If the diet was deprived of fat, however, this polypeptide species, with an estimated molecular weight of 80 000, decreased to a level slightly lower than normal. On the basis of the electron micrographic criteria, the high-fat diet provoked a marked proliferation of hepatic peroxisomes.