24(S)‐hydroxycholesterol is actively eliminated from neuronal cells by ABCA1

High cholesterol turnover catalyzed by cholesterol 24‐hydroxylase is essential for neural functions, especially learning. Because 24(S)‐hydroxycholesterol (24‐OHC), produced by 24‐hydroxylase, induces apoptosis of neuronal cells, it is vital to eliminate it rapidly from cells. Here, using differentiated SH‐SY5Y neuron‐like cells as a model, we examined whether 24‐OHC is actively eliminated via transporters induced by its accumulation. The expression of ABCA1 and ABCG1 was induced by 24‐OHC, as well as TO901317 and retinoic acid, which are ligands of the nuclear receptors liver X receptor/retinoid X receptor (LXR/RXR). When the expression of ABCA1 and ABCG1 was induced, 24‐OHC efflux was stimulated in the presence of high‐density lipoprotein (HDL), whereas apolipoprotein A‐I was not an efficient acceptor. The efflux was suppressed by the addition of siRNA against ABCA1, but not by ABCG1 siRNA. To confirm the role of each transporter, we analyzed human embryonic kidney 293 cells stably expressing human ABCA1 or ABCG1; we clearly observed 24‐OHC efflux in the presence of HDL, whereas efflux in the presence of apolipoprotein A‐I was marginal. Furthermore, the treatment of primary cerebral neurons with LXR/RXR ligands suppressed the toxicity of 24‐OHC. These results suggest that ABCA1 actively eliminates 24‐OHC in the presence of HDL as a lipid acceptor and protects neuronal cells.     

Induction of Anti-influenza Immunity by Modified Green Fluorescent Protein (GFP) Carrying Hemagglutinin-derived Epitope Structure

The development of vaccination methods that can overcome the emergence of new types of influenza strains caused by escape mutations is desirable to avoid future pandemics. Here, a novel type of immunogen was designed that targeted the conformation of a highly conserved region of influenza A virus hemagglutinin (HA) composed of two separate sequences that associate to form an anti-parallel β-sheet structure. Our previous study identified this β-sheet region as the structural core in the epitope of a characteristic antibody (B-1) that strongly neutralizes a wide variety of strains within the H3N2 serotype, and therefore this β-sheet region was considered a good target to induce broadly reactive immunity against the influenza A virus. To design the immunogen, residues derived from the B-1 epitope were introduced directly onto a part of enhanced green fluorescent protein (EGFP), whose surface is mostly composed of β-sheets. Through site-directed mutagenesis, several modified EGFPs with an epitope-mimicking structure embedded in their surface were prepared. Two EGFP variants, differing from wild-type (parental) EGFP by only five and nine residues, induced mice to produce antibodies that specifically bind to H3-type HA and neutralize H3N2 virus. Moreover, three of five mice immunized with each of these EGFP variants followed by a booster with equivalent mCherry variants acquired anti-viral immunity against challenge with H3N2 virus at a lethal dosage. In contrast to conventional methods, such as split HA vaccine, preparation of this type of immunogen requires less time and is therefore expected to be quickly responsive to newly emerged influenza viral strains.     

The Bcl-2 Homology Domain 3 (BH3)-only Proteins Bim and Bid Are Functionally Active and Restrained by Anti-apoptotic Bcl-2 Family Proteins in Healthy Liver

An intrinsic pathway of apoptosis is regulated by the B-cell lymphoma-2 (Bcl-2) family proteins. We previously reported that a fine rheostatic balance between the anti- and pro-apoptotic multidomain Bcl-2 family proteins controls hepatocyte apoptosis in the healthy liver. The Bcl-2 homology domain 3 (BH3)-only proteins set this rheostatic balance toward apoptosis upon activation in the diseased liver. However, their involvement in healthy Bcl-2 rheostasis remains unknown. In the present study, we focused on two BH3-only proteins, Bim and Bid, and we clarified the Bcl-2 network that governs hepatocyte life and death in the healthy liver. We generated hepatocyte-specific Bcl-xL- or Mcl-1-knock-out mice, with or without disrupting Bim and/or Bid, and we examined hepatocyte apoptosis under physiological conditions. We also examined the effect of both Bid and Bim disruption on the hepatocyte apoptosis caused by the inhibition of Bcl-xL and Mcl-1. Spontaneous hepatocyte apoptosis in Bcl-xL- or Mcl-1-knock-out mice was significantly ameliorated by Bim deletion. The disruption of both Bim and Bid completely prevented hepatocyte apoptosis in Bcl-xL-knock-out mice and weakened massive hepatocyte apoptosis via the additional in vivo knockdown of mcl-1 in these mice. Finally, the hepatocyte apoptosis caused by ABT-737, which is a Bcl-xL/Bcl-2/Bcl-w inhibitor, was completely prevented in Bim/Bid double knock-out mice. The BH3-only proteins Bim and Bid are functionally active but are restrained by the anti-apoptotic Bcl-2 family proteins under physiological conditions. Hepatocyte integrity is maintained by the dynamic and well orchestrated Bcl-2 network in the healthy liver.     

Interaction of dimeric horse cytochrome c with cyanide ion

We have previously shown that methionine–heme iron coordination is perturbed in domain-swapped dimeric horse cytochrome c. To gain insight into the effect of methionine dissociation in dimeric cytochrome c, we investigated its interaction with cyanide ion. We found that the Soret and Q bands of oxidized dimeric cytochrome c at 406.5 and 529 nm redshift to 413 and 536 nm, respectively, on addition of 1 mM cyanide ion. The binding constant of dimeric cytochrome c and cyanide ion was obtained as 2.5 × 10⁴ M^?1. The Fe–CN and C–N stretching (ν _Fe–CN and ν _CN) resonance Raman bands of CN^?-bound dimeric cytochrome c were observed at 443 and 2,126 cm^?1, respectively. The ν _Fe–CN frequency of dimeric cytochrome c was relatively low compared with that of other CN^?-bound heme proteins, and a relatively strong coupling between the Fe–C–N bending and porphyrin vibrations was observed in the 350–450-cm^?1 region. The low ν _Fe–CN frequency suggests weaker binding of the cyanide ion to dimeric cytochrome c compared with other heme proteins possessing a distal heme cavity. Although the secondary structure of dimeric cytochrome c did not change on addition of cyanide ion according to circular dichroism measurements, the dimer dissociation rate at 45 °C increased from (8.9 ± 0.7) × 10^?6 to (3.8 ± 0.2) × 10^?5 s^?1, with a decrease of about 2 °C in its dissociation temperature obtained with differential scanning calorimetry. The results show that diatomic ligands may bind to the heme iron of dimeric cytochrome c and affect its stability.     

Changes in 1-Aminocyclopropane-1-carboxylic Acid Content and Ethylene Production in Hiproly Barley Callus during Differentiation

During differentiation after auxin withdrawal, the change in the ethylene production of Hiproly barley callus paralleled the change in 1-aminocyclopropane-1-carboxylic acid (ACC) content. The levels of ACC and ethylene production decreased rapidly, and then increased in Hiproly barley callus.

Aminooxyacetic acid (AOA) prevented the ACC and ethylene production of the callus. Moreover, aminoisobutyric acid (AIB) also inhibited the ethylene production, but did not prevent the ACC synthesis of the callus. On the other hand, methylglyoxal-bis(guanylhydrazone) (MGBG) greatly enhanced the ACC and ethylene production. Formation of adventitious roots in Hiproly barley callus was enhanced by the cultivation in the medium containing AIB or AOA. However, differentiation of the callus was strongly inhibited by MGBG.

Thus, prevention of ethylene production may be significant for differentiation of Hiproly barley callus.     

Synthesis of 5-Alkyl-5-methyl-2-hydroxy-2-cyclopentenones

A hydrogen bacterium strain, N34, and its oxygen-resistant segregant strain, Y38, were subjected to a taxonomical study. Since both strains were capable of N₂-fixation, N₂-fixing facultative hydrogen autotrophs listed in “Bergey’s Manual of Systematic Bacteriology” were used for comparison. Both strains produced a water-insoluble carotenoid pigment, zeaxanthin dirhamnoside, indicating that both should be classified into the genus Xanthobacter. Then, the differential characteristics of the two species of the genus Xanthobacter, X. autotrophicus and X. flavus, were investigated as to both strains. The vitamin requirement, the sensitivity to oxygen under autotrophic conditions, the inducibility of hydrogenase, the substrate range of carbohydrates and N₂-fixing growth characteristics of both strains were almost completely opposite to those of X. flavus. Moreover, both strains coincided exactly with X. autotrophicus in morphological and other physiological characteristics. From these results both strains were identified as Xanthobacter autotrophicus.     

Crystallization and Properties of N-Benzoylglycine Amidohydrolase from Pseudomonas putida

N-Benzoylgiycine amidohydrolase (hippurate hydrolase EC 3.5.1.32), which catalyzes the hydrolysis of hippuric acid to benzoic acid and glycine, was found in a cell-free extract of Pseudomonas putida C692-3 grown on a medium containing hippuric acid. The enzyme was purified from the extract by ammonium sulfate fractionation and column chromatographies on DEAE-cellulose, DEAE-Sephadex A-50, hydroxyapatite, and Sepharose CL-6B. The enzyme was finally crystallized. The crystalline enzyme was almost homogeneous on electrophoresis. The enzyme had a molecular weight of about 170,000 and consisted of four subunits identical in molecular weight (approximately 42,000). The enzyme hydrolyzed N-benzoylglycine most rapidly, and N-benzoyl-l-alanine and N-benzoyl-l-aminobutyric acid. The Km value for these substrates were 0.72 mm, 0.87 mm, and 0.87mm, respectively. The optimum pH of the enzyme reaction was 7.0 to 8.0 and the enzyme was stable from pH 6.0 to 8.0.     

Proteins Released from Myofibrils by CASF (Ca2+-activated sarcoplasmic factor) and Trypsin

The nature of the soluble proteins and peptides released from myofibrils by treatment with CASF (Ca²⁺-activated sarcoplasmic factor) was investigated by using Polyacrylamide gel electrophoresis in both a nondenaturing and a denaturing (sodium dodecyl sulfate=SDS) solvent and by using gel permeation chromatography on Sepharose 6B. Both CASF and trypsin treatment cause removal of Z-disks before causing other ultrastructurally detectable degradation of myofibrils. CASF treatment of myofibrils releases a protein that is identical to α-actinin, one of the known components of the Z-disk, on the basis of mobility in Polyacrylamide gel electrophoresis in a nondenaturing solvent and in SDS and on the basis of elution from gel permeation columns. Trypsin treatment of myofibrils releases a number of smaller molecular weight products that cannot be identified with any of the known myofibrillar proteins. Hence, CASF seems to remove Z-disks from myofibrils by means of a very specific proteolytic activity that releases α-actinin without extensively degrading it. Trypsin, on the other hand, probably seems to remove Z-disks by degrading α-actinin to peptides.     

Syntheses and Biological Activities of New Plant Growth Inhibitors Structurally Related to Abscisic Acid

Several analogs of abscisic acid (ABA) were prepared and their biological activities were assayed. Among the compounds tested, 5-(l, 2-epoxy-2, 6, 6-trimethyl-l-cyclohexyl)-, 5-(l-hydroxy-2, 6, 6-trimethyl-2-cyclohexen-l-yl)- and 5-(l-hydroxy-2-methylene-6, 6-dimethyl-l-cyclohexyl)-3-methyl-cis, trans-2, 4-pentadienoic esters (V, IX, XXIII and XXV) were found to be potent plant growth inhibitors. Their activities were superior or comparable to that of ABA.