A morphological study and hybridization analysis of Caloglossa leprieurii (Ceramiales, Rhodophyta) from Japan, Singapore and Australia

Morphological and hybridization experiments were performed on Caloglossa leprieurii (Montagne) J. Agardh collected from Japan, Singapore and Australia in order to evaluate taxonomic characters of this species. Within C. leprieurii at least four mating groups were recognized from the Indo-Pacific region. These mating groups could be characterized by the blade width at the inter-node and the cell-row numbers on the opposite side derived from the first axial cell at the main axis, though these properties showed a certain variability even in the same plant under both field and culture conditions. The phylogenetic relationship and geographic distribution of the four mating groups are discussed.     

Enantioselective dehydrogenation of β-hydroxysilanes by horse liver alcohol dehydrogenase with a novel in-situ NAD+ regeneration system

Dehydrogenation of 2-trimethylsilyl-1-propanol (1) was carried out with horse liver alcohol dehydrogenase (HLADH, EC 1.1.1.1). It was found that the hydrogenation of 1 proceeded enantioselectively with only HLADH and a catalytic amount of NAD⁺ due to in-situ NAD⁺ regeneration based on a specific property of -carbonylsilanes. That is, (+)-1 was enantioselectively dehydrogenated by HLADH to 2-trimethylsilyl-1-propanal, which was spontaneously degraded by addition of water into trimethylsilanol and n-propanal. Then, NAD⁺ was regenerated through HLADH-catalyzed reduction of n-propanal to n-propanol. On the other hand, dehydrogenation of the carbon analogue of 1 was negligible with a catalytic amount of NAD⁺, indicating that the in-situ NAD⁺ regeneration was not available without the specific property of organosilicon compounds. Other primary -hydroxysilanes having different substituents on the chiral center or on the silicon atom were also found to serve as substrates in enantioselective dehydrogenation by HLADH with this novel NAD⁺ regeneration system. Chiral recognition of HLADH toward primary alcohols is also discussed.     

3-Aminobenzamide,a potent inhibitor of poly (ADP-ribose) polymerase,causes a rapid death of HL-60 cells cultured in serum-free medium

HL-60 cells transferred from serum-supplemented to serum-free culture medium initially bound to culture plate tightly and then released from the plate on increasing the culture time and resumed exponential growth after about 8 h lag. At the initial stage of the culture, the cells became extremely sensitive to 3-aminobenzamide, a potent inhibitor of poly (ADP-ribose) polymerase, and, at 1 mM, 80 to 90% of the cells were lysed within 20 h, whereas the inhibitor was totally ineffective on the cell growth in serum-supplemented medium at the concentration. Non-inhibitory analogs of the inhibitor were ineffective. Assay of poly(ADP-ribose) polymerase activity in permeable cells indicated that a transient activation of the enzyme occurred during the culture in serum-free medium (the maximum activation was observed at 8 h of the culture). The cells conditioned in serum-free medium for 24 h acquired significant resistancy to the inhibitor. A low concentration of fibronectin (5 to 10/ml) and a relatively high concentration of bovine serum albumin (0.5 to 1 mg/ml) effectively blocked the cell attachment to plate and also the 3-aminobenzamide-induced cell lysis. These results suggest that poly(ADP-ribose) polymerase is involved in a process essential for HL-60 cells to adapt to a serumdeprived growth condition.     

The Eiken Latex test for detection of a cryptococcal antigen in cryptococcosis

A latex agglutination test for cryptococcal antigen, the Eiken Latex test (Eiken, Tokyo, Japan), was compared with a monoclonal antibody-based agglutination assay, Pastorex^® Cryptococcus (Diagnostics Pasteur, Marneur-la-Coquette, France). In a murine model of disseminated cryptococcosis, the kinetics of the antigen titers by the Eiken Latex were similar to those by the Pastorex^® Cryptococcus, but sensitivity was much higher. In HIV-negative patients with pulmonary cryptococcosis, a cryptococcal antigen was detected in 6 of 10 patients by the Eiken Latex test and in only 3 of those patients by the Pastorex^® Cryptococcus. The results indicate that the Eiken Latex is more sensitive for the detection of the cryptococcal antigen, even in non-disseminated cryptococcosis. The sensitivity and specificity of the Eiken Latex were examined using 195 sera from 25 patients with pulmonary cryptococcosis and 170 patients with non-cryptococcosis. The cutoff value of 1:8 showed a sensitivity of 76% (19/25) and a specificity of 98.9% (168/170).     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

Sialosylcholesterol Effects on Reconstitution of Microfilament and Glia Filament

Abstract: The effects of α-sialosylcholesterol (α-SC) on formation of either microfilament or glia filament of rat astrocytes were investigated using a reconstitution system. Polymerization of the depolymerized microfilament preparation that had been extracted from a crude cytoskeletal fraction of rat astrocytes, in the presence of 100 m M KCI and 10 m M MgCI₂, was suppressed in a dose-dependent manner by α-SC. α-SC inhibited polymerization of G-actin in a similar manner. The intensity of a-SC inhibition of G- actin polymerization was as great as that of microfilament polymerization, suggesting that the inhibition of microfilament polymerization by α-SC was due to the direct action of α-SC on actin, the main component of microfilament. α-SC depolymerized partly the polymerized microfilament preparation, which resembled F-actin (microfilament-like filaments). α-SC suppressed, in a dose-dependent manner, polymerization of a glia filament preparation that had been extracted from astrocyte cytoskeletons in the presence of phalloidin. An increase in the amount of added α-SC (up to 15 n M ) decreased the amount of the larger glia filament-like filaments, which were 10 nm thick and centrifuged down at 16,000 g for 30 min, and increased that of smaller ones precipitated only after centrifugation at 100,000 g for 1 h. The lower the concentration of the depolymerized glia filament extract, the greater was the inhibition by α-SC of the polymerization. α-SC repressed polymerization of vimentin, the dominant component of glia filament. Vimentin polymerization was more strongly inhibited by α-SC than polymerization of glia filament was. The findings suggested that α-SC suppressed polymerization of glia filament through a direct action on vimentin and that the glia filament-associated proteins increased its structural stability in the presence of α-SC.     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Flower-Inducing Activity of Exogenous Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Rich Medium

Flower-inducing activity of lysine was examined in Lemna paucicostata151, a weakly responsive short-day plant, cultured on nitrogen-richmedium under long-day conditions (continuous light). Lemna paucicostata151 was homogenized in a solution of lysine and the homogenatewas centrifuged. The supernatant (lysine-containing extract)was added to nitrogen-rich medium after passage through a membranefilter to give various concentrations of lysine in the medium.Flowering was induced in plants grown for six days on mediumthat contained lysine at concentrations above 0.25 µM.In plants grown on medium that contained 1 µM lysine,a significant flowering response was observed on the fourthday of culture. However, the flower-inducing activity of lysinedisappeared when the lysine-containing extract was added tothe medium and the medium was then autoclaved, suggesting thatthe active principle is unstable to autoclaving. Among derivativesof lysine tested, lysine hydroxamate had the highest flower-inducingactivity and lysyl lysine had almost same activity as that oflysine. When added to the medium without homogenization withplant material, lysine and lysyl lysine had flower-inducingactivity but lysine hydroxamate did not induce flowering. (Received April 26, 1993; Accepted November 8, 1993)