Binding-site Analysis of the Ether Linkages between Lignin and Hemicelluloses in Lignin–Carbohydrate Complexes by DDQ-Oxidation

Lignin-carbohydrate complexes (LCC; nor-C-1-M, com-C-1-A) isolated from normal and compression woods of Pinus densiflora were hydrolyzed with two types of cellulase preparations, and the hydrolyzates formed were fractionated by adsorption chromatography on polyvinyl gel into water-soluble materials and LCC fragments. To elucidate the binding sites between the lignin and carbohydrate, the cellulase-degraded LCC fragments were subjected to acetylation, and then oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which was confirmed to oxidatively cleave the benzyl ether linkages between the lignin and carbohydrate. The DDQ-oxidized fraction was then methylated by the method of Prehm, hydrolyzed, reduced and acetylated. A GC-MS analysis of the methylated sugar revealed that alditol acetates from 6-O-methyl mannose, 6-O-methyl galactose, 6-O-methyl glucose and a small amount of their 2-O- or 3-O-methyl isomers existed in both methylated fractions. 2-O-Methyl xylose and 3-O-methyl xylose were also identified in the fraction from the acidic LCC (com-C-1-A). These results led to the conclusion that acetylglucomannan and β-1,4-galactan were preferably bound to the lignin at C-6 position of the hexoses, and that arabinoglucuronoxylan did likewise at the C-2 and C-3 positions of xylose units.     

Measurement of resistant starch content in cooked rice and analysis of gelatinization and retrogradation characteristics

Digestion-resistant starch (RS) has many physiologic functions. The RS content is measured by enzymatically degrading flour samples according to the method of the Association of Official Analytical Chemists. Experiments have been performed with wheat, corn, and other grains, but there are no data for cooked rice grains in the form ingested by humans. Thus, we investigated a method to measure RS that is suitable for cooked rice grains using rice cultivars that are reported to differentially increase postprandial blood glucose in humans. Using a method for cooking individual rice grains and optimized enzyme reaction conditions, we established an RS measurement method. We also found that the amylopectin crystal condition affects the RS content measured using our method.     

Roseoflavin Formation by Streptomyces davawensis

We previously found that one of the pharmacological effects of N-tert-butyl-α-phenylnitrone (PBN) is the release of nitric oxide (NO) under oxidative conditions. However, to confirm this hypothesis in vivo, NO released from PBN must be distinguished from NO produced in biological systems, and therefore we undertook the synthesis of PBN using labeled ¹⁵N to identify its corresponding ¹⁵NO in vivo. The properties were examined with an ESR spectrometer. To synthesize ¹⁵N-PBN, the starting material, ammonium-¹⁵N chloride, was converted to 2-amino-¹⁵N-2-methylpropane, oxidized to 2-methyl-2-nitropropane-¹⁵N, and finally reacted with benzaldehyde to give ¹⁵N-PBN. The final product was purified by repeated sublimation. With ferrous sulfate-methyl glucamine dithiocarbamate complex, Fe (MGD)₂, as a trapping agent to measure the NO levels of ¹⁵N-PBN or ¹⁴N-PBN in vitro, the peak intensity of ¹⁵NO[Fe(MGD)₂] was over 50% stronger than that of ¹⁴NO[Fe(MGD)₂], and that ¹⁵NO and ¹⁴NO had the corresponding two-and three line hyperfine structures due to their nuclear spin quantum numbers. Subsequently, the ESR spectrum of ¹⁵NO derived from ¹⁵N-PBN was significantly different than that of lipopolysaccharide (LPS)-induced NO, which was derived from biological cells, and therefore we have demonstrated the possibility to distinguish ¹⁵NO from PBN and ¹⁴NO generated from cells. These results suggested that ¹⁵N-PBN is a useful molecule, not only as a spin-trapping agent, but also as an NO donor to explore the pharmacological mechanisms of PBN in vivo.     

Role of progesterone receptors during postpartum estrus in rats

We studied the role of progesterone receptor (PR) in the display of female sexual behavior during postpartum estrus in rats. Adult female rats were treated with the PR antagonist, RU486 (1.25 and 5 mg), 3 h after parturition and sexual behavior was evaluated throughout the first postpartum day. Estradiol and progesterone serum levels changed during the first 24 h postpartum. The highest estradiol and progesterone levels were found at 9 and 12 h postpartum, respectively. The predominant PR isoform in the hypothalamus and the preoptic area was PR-A during postpartum day. The content of PR-A increased at 6 h postpartum in the hypothalamus and the preoptic area, and decreased in both regions at 9 h. PR-B content only increased in the preoptic area at 12 h postpartum. The highest display of lordotic and proceptive behaviors were found at 12 h postpartum. The treatment with 1.25 and 5 mg of RU486 respectively reduced lordosis by 61% and 92% at 12 h postpartum. These results suggest that PR is essential in the display of postpartum estrus in rats.     

Impact of self-association on function of apolipoprotein A-I

Self-association is an inherent property of the lipid-free forms of several exchangeable apolipoproteins, including apolipoprotein A-I (apoA-I), the main protein component of high density lipoproteins (HDL) and an established antiatherogenic factor. Monomeric lipid-free apoA-I is believed to be the biologically active species, but abnormal conditions, such as specific natural mutations or oxidation, produce an altered state of self-association that may contribute to apoA-I dysfunction. Replacement of the tryptophans of apoA-I with phenylalanines (ΔW-apoA-I) leads to unusually large and stable self-associated species. We took advantage of this unique solution property of ΔW-apoA-I to analyze the role of self-association in determining the structure and lipid-binding properties of apoA-I as well as ATP-binding cassette A1 (ABCA1)-mediated cellular lipid release, a relevant pathway in atherosclerosis. Monomeric ΔW-apoA-I and wild-type apoA-I activated ABCA1-mediated cellular lipid release with similar efficiencies, whereas the efficiency of high order self-associated species was reduced to less than 50%. Analysis of specific self-associated subclasses revealed that different factors influence the rate of HDL formation in vitro and ABCA1-mediated lipid release efficiency. The α-helix-forming ability of apoA-I is the main determinant of in vitro lipid solubilization rates, whereas loss of cellular lipid release efficiency is mainly caused by reduced structural flexibility by formation of stable quaternary interactions. Thus, stabilization of self-associated species impairs apoA-I biological activity through an ABCA1-mediated mechanism. These results afford mechanistic insights into the ABCA1 reaction and suggest self-association as a functional feature of apoA-I. Physiologic mechanisms may alter the native self-association state and contribute to apoA-I dysfunction.     

Birth of cloned miniature pigs derived from somatic cell nuclear transferred embryos activated by ultrasound treatment

The present study was carried out to determine (1) the optimal duty cycle of ultrasound for activation of pig oocytes and cloned embryos derived from miniature pig fetal fibroblasts and (2) whether cloned embryos can develop to term following activation by ultrasound stimulation. When oocytes were exposed to ultrasound with 20% or 30% duty cycle, the blastocyst formation rates were significantly (P < 0.05) higher than that of oocytes exposed to ultrasound with 10% duty cycle. In contrast, the blastocyst formation rate of cloned embryos decreased as the duty cycle of ultrasound increased; the value of embryos exposed to ultrasound with 10% duty cycle was significantly (P < 0.05) higher than that of embryos exposed to ultrasound with 50% duty cycle. When cloned embryos exposed to ultrasound with 10% duty cycle were transferred into the oviducts of two recipient gilts to assess their development in vivo, the pregnancy of one of the gilts was maintained to term and two piglets were delivered via Cesarean section. The results of the present study showed that (1) although the duty cycle of ultrasound affects in vitro development after activation of both pig oocytes and miniature pig cloned embryos, the optimal duty cycle is different between them and (2) miniature pig cloned embryos have the ability to develop into piglets after activation by ultrasound stimulation.     

Deficiency in the Lipid Exporter ABCA1 Impairs Retrograde Sterol Movement and Disrupts Sterol Sensing at the Endoplasmic Reticulum

Cellular cholesterol homeostasis involves sterol sensing at the endoplasmic reticulum (ER) and sterol export from the plasma membrane (PM). Sterol sensing at the ER requires efficient sterol delivery from the PM; however, the macromolecules that facilitate retrograde sterol transport at the PM have not been identified. ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol and phospholipid export to apolipoprotein A-I for the assembly of high density lipoprotein (HDL). Mutations in ABCA1 cause Tangier disease, a familial HDL deficiency. Several lines of clinical and experimental evidence suggest a second function of ABCA1 in cellular cholesterol homeostasis in addition to mediating cholesterol efflux. Here, we report the unexpected finding that ABCA1 also plays a key role in facilitating retrograde sterol transport from the PM to the ER for sterol sensing. Deficiency in ABCA1 delays sterol esterification at the ER and activates the SREBP-2 cleavage pathway. The intrinsic ATPase activity in ABCA1 is required to facilitate retrograde sterol transport. ABCA1 deficiency causes alternation of PM composition and hampers a clathrin-independent endocytic activity that is required for ER sterol sensing. Our finding identifies ABCA1 as a key macromolecule facilitating bidirectional sterol movement at the PM and shows that ABCA1 controls retrograde sterol transport by modulating a certain clathrin-independent endocytic process.     

Systematics of the Hipposideros turpis complex and a description of a new subspecies from Vietnam

• 1 Hipposideros turpis is traditionally known as a species composed of three subspecies, H. t. turpis, H. t. alongensis and H. t. pendleburyi, distributed disjunctly in south‐west Japan, north‐east Vietnam and south‐west Thailand, respectively. Prior to the present study, the systematic status of forms within the species remained unclear.
• 2 Using morphological (external, bacular, cranial and dental characters), genetic and echolocation data, we demonstrate that turpis, alongensis and pendleburyi represent three distinct species, and that these species are endemic to Japan, Vietnam and Thailand, respectively. They are very distinct genetically and do not even form a monophyletic group.
• 3 We also prove that H. alongensis is composed of two subspecies, H. a. alongensis and H. a. sungi. The latter subspecies is described as new to science. To date, H. a. alongensis appears to be restricted to the Cat Ba Island of Cat Ba National Park, west Ha Long Bay, whereas H. a. sungi ssp. nov. is known from three localities in mainland northeast Vietnam. These two subspecies are distinguished by body size, molecular data and the frequency of the constant‐frequency component of their echolocation signals.

     

Effect of the oral intake of yogurt containing Bifidobacterium longum BB536 on the cell numbers of enterotoxigenic Bacteroides fragilis in microbiota

Enterotoxigenic Bacteroides fragilis (ETBF) strains have been suggested to be associated with acute and persistent diarrheal disease, inflammatory bowel disease and colorectal cancer, although further epidemiological studies are needed for clarification. Here, a pilot study was performed to examine the effect of the oral administration of yogurt supplemented with a probiotic strain on the cell numbers of fecal ETBF in a healthy population. Among 420 healthy adults, 38 subjects were found to be ETBF carriers, giving a prevalence of approximately 9%. Among them, 32 subjects were enrolled in an open, randomized, parallel-group study to ingest yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536 (BB536Y group), for 8 weeks, with milk provided to the control group (milk group). The cell numbers of ETBF and the dominant species of the B. fragilis group were measured by a quantitative PCR method. Compared with the baseline values, there was a significant decrease in the cell number of ETBF at week 8 in the BB536Y group but not in the milk group. Linear mixed models analysis for longitudinal data revealed a significant difference in the changes of ETBF cell number between the two groups during the intervention phase. These results imply the potential of probiotic yogurt for eliminating ETBF in the microbiota, but its clinical significance needs to be evaluated in the future. This is the first report of a possible effect of probiotic intake on ETBF in the microbiota.