Influence of State of Mixing of the Sample on Total Absorbed Energy in Microwave Cooking

The effects of the state of mixing on total absorbed energy (P) in microwave cooking were investigated. When water and oil were heated independently, both of P were almost equal and the effect of the dielectric loss factor (ε″) on P was negligible. P of a two-layer sample was almost equal to that of the water component alone and was affected by ε″. P of an emulsion sample was almost constant and equal to that of water alone whose volume was the same as the volume of the sample. The difference between P of a two-layer sample and that of an emulsion sample was thought to be affected by the increase in the water-oil interface area, and it was found that P of the water-oil mixture is influenced by the state of mixing.     

The Inhibition of α-Amylase by Tea Polyphenols

The inhibition of α-amylase from human saliva by polyphenolic components of tea and its specificity was investigated in vitro. Four kinds of green tea catechins, and their isomers and four kinds of their dimeric compounds (theaflavins) produced oxidatively during black tea production were isolated. They were (?)-epicatechin (EC), (?)-epigallocatechin (EGC), (?)-epicatechin gallate (ECg), (?)-epigallocatechin gallate (EGCg), (?)-catechin (C), (?)-gallocatechin (GC), (?)-catechin gallate (Cg), (?)-gallocatechin gallate (GCg), theaflavin (TF1), theaflavin monogallates (TF2A and TF2B), and theaflavin digallate (TF3). Among the samples tested, EC, EGC, and their isomers did not have significant effects on the activity of α-amylase. All the other samples were potent inhibitors and the inhibitory effects were in the order of TF3>TF2A>TF2B>TFl>Cg> GCg > ECg > EGCg. The inhibitory patterns were noncompetitive except for TF3.     

Expression of Mature Human Interleukin 2 and Its Derivatives in Escherichia coli and Comparison of Their Biological Activity in Vitro

A mature human interleukin 2 (hIl-2) and its derivatives that lacked the N-terminal portion were expressed in Escherichia coli under the control of the phage λ P_L promoter. They accumulated in the form of insoluble inclusion bodies and accounted for about 30% of the total cellular protein. The mature hIl-2 and its derivatives were further purified and their in vitro biological activity was compared in an Il-2 microassay. The results suggested that the hIl-2 derivatives without the N- terminal three or five amino acids were as active as intact hIl-2 and that those without the N- terminal eight or nine amino acids were less active than the intact form.     

Substrate Specificity and Some Properties of Crystalline Mold Maltase

The substrate specificity of crystalline mold maltase was investigated.

The enzyme acts upon various α-heteroglucosides or saccharides. Aryl-α-glucosides were hydrolyzed much faster than alkyl-α-glucosides. The enzyme acts on the maltose derivatives whose reducing groups have been masked. But among glucosylfructoses turanose, maltulose and isomaltulose were attacked with a slow rate while the enzyme was quite inert to sucrose. Malto- and isomalto-oligosaccharides were also hydrolyzed and the enzyme ceased its action at seven to eight units of hexose in both series of oligosaccharides.

The opt. pH range of Takamaltase was 4.2~4.6 and opt. temp., 50~55°C. Cu⁺⁺ and Hg⁺⁺ strongly inhibited the enzyme activity but other metal ions tested had no effects. It is suggested that the enzyme is not a sulfhydryl enzyme because of the lack of effects of SH-reagents on the activity.     

Albino (al) is a tetrahydrobiopterin (BH4)-deficient mutant of the silkworm Bombyx mori

Albino (al) is a lethal mutant of Bombyx mori that exhibits a colourless cuticle after the first ecdysis and dies without feeding on mulberry. Previous studies have indicated that sclerotisation was insufficient because of defective phenylalanine and tyrosine metabolism in albino larvae. However, the genetic mechanism underlying the albino phenotype has not been determined. Dopamine plays a central role in insect cuticle colouration and sclerotisation. The pathway for dopamine biosynthesis from phenylalanine involves phenylalanine hydroxylase (PAH; EC 1.14.16.1) and tyrosine hydroxylase (TH; EC 1.14.16.2). Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases, including PAH and TH. Thus, BH4 is indispensable for cuticle colouration and sclerotisation. Here we report on identifying mutations in the gene that encodes for the Bombyx homolog of 6-pyruvoyl-tetrahydropterin synthase (PTS) which is involved in the biosynthesis of BH4, in 2 strains with different al alleles. In strain a60 (al), a transposable element was inserted in exon 2 of BmPTS. In strain a61 (al²), an 11-bp deletion was identified in the exon 2 region of BmPTS. After oral administration of BH4 to the al² larvae, the survival rate was effectively increased and the larval integument was pigmented. These results indicated that BmPTS was responsible for the albino mutants of B. mori. We conclude that (i) a mutation in BmPTS leads to an insufficient supply of BH4 and results in defective dopamine biosynthesis and (ii) lack of dopamine results in cuticle colouration and sclerotisation failure. Lemon (lem) is a BH4-deficient mutant. It has been reported that de novo synthesis of zygotic BH4 was indispensable for viability of the embryo in eggs laid by lem (lem/lem^l) females. We found that lem/lem, al²/al² larvae produced by lem (lem/lem) females were viable during the first instar stage, suggesting that al²/al² embryo could synthesis BH4 by using maternally transmitted BmPTS.     

A novel and mild isolation procedure of chlorosomes from the green sulfur bacterium Chlorobaculum tepidum

In this article, we developed a new and mild procedure for the isolation of chlorosomes from a green sulfur bacterium Chlorobaculum tepidum. In this procedure, Fenna-Matthews-Olson (FMO) protein was released by long cold treatment (6°C) of cells under the presence of a chaotrope (2?M NaSCN) and 0.6?M sucrose. Chlorosomes were released by an osmotic shock of the cold-treated cells after the formation of spheroplasts without mechanical disruption. Chlorosomes were finally purified by a sucrose step-wise density gradient centrifugation. We obtained two samples with different density (20 and 23% sucrose band, respectively) and compared them by SDS-PAGE, absorption spectroscopy at 80?K, fluorescence and CD spectroscopy at room temperature. Cells whose absorption maximum was longer than 750?nm yielded higher amount of the 20% sucrose fraction than those having an absorption maximum shorter than 750?nm.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Amelioration of D-galactosamine-induced acute liver injury in rats by dietary supplementation with betaine derived from sugar beet molasses

The effects of betaine supplementation on D-galactosamine-induced liver injury were examined in terms of hepatic and serum enzyme activities and of the levels of glutathione and betaine-derived intermediates. The rats induced with liver injury showed marked increases in serum enzyme activity, but those receiving dietary supplementation of 1% betaine showed enzyme activity levels similar to a control group without liver injury. Administration of betaine also increased both hepatic and serum glutathione levels, even following D-galactosamine injection. The activity of glutathione-related enzymes was markedly decreased following injection of D-galactosamine, but remained comparable to that of the control group in rats receiving 1% betaine. The concentrations of hepatic S-adenosyl methionine and cysteine showed similar trends to that observed for hepatic glutathione levels. These results indicate that 1% betaine has a hepatoprotective effect by increasing hepatic and serum glutathione levels along with glutathione-related enzyme activities in rats.     

Identification of 4-quinolone derivatives as inhibitors of reactive oxygen species production from human umbilical vein endothelial cells

Oxidative stress is widely recognized as being associated with a number of disorders, including metabolic dysfunction and atherosclerosis. A series of substituted 4-quinolone derivatives were prepared and evaluated as inhibitors of reactive oxygen species (ROS) production from human umbilical vein endothelial cells (HUVECs). One compound in particular, 2-({[4-(3-hydroxy-3-methylbutoxy)pyridin-2-yl]oxy}methyl)-3-methylquinolin-4(1H)-one (25b), inhibited ROS production from HUVECs with an IC(50) of 140 nM. This compound also exhibited low CYP2D6 inhibitory activity, high aqueous solubility, and good in vitro metabolic stability. An in vivo pharmacokinetic study of this compound in SD rats revealed high oral bioavailability and a long plasma half-life.     

Two distinct amyloid beta-protein (Abeta) assembly pathways leading to oligomers and fibrils identified by combined fluorescence correlation spectroscopy, morphology, and toxicity analyses

Nonfibrillar assemblies of amyloid β-protein (Aβ) are considered to play primary roles in Alzheimer disease (AD). Elucidating the assembly pathways of these specific aggregates is essential for understanding disease pathogenesis and developing knowledge-based therapies. However, these assemblies cannot be monitored in vivo, and there has been no reliable in vitro monitoring method at low protein concentration. We have developed a highly sensitive in vitro monitoring method using fluorescence correlation spectroscopy (FCS) combined with transmission electron microscopy (TEM) and toxicity assays. Using Aβ labeled at the N terminus or Lys(16), we uncovered two distinct assembly pathways. One leads to highly toxic 10-15-nm spherical Aβ assemblies, termed amylospheroids (ASPDs). The other leads to fibrils. The first step in ASPD formation is trimerization. ASPDs of ～330 kDa in mass form from these trimers after 5 h of slow rotation. Up to at least 24 h, ASPDs remain the dominant structures in assembly reactions. Neurotoxicity studies reveal that the most toxic ASPDs are ～128 kDa (～32-mers). In contrast, fibrillogenesis begins with dimer formation and then proceeds to formation of 15-40-nm spherical intermediates, from which fibrils originate after 15 h. Unlike ASPD formation, the Lys(16)-labeled peptide disturbed fibril formation because the Aβ(16-20) region is critical for this final step. These differences in the assembly pathways clearly indicated that ASPDs are not fibril precursors. The method we have developed should facilitate identifying Aβ assembly steps at which inhibition may be beneficial.