Relictithismia kimotsukiensis, a new genus and species of Thismiaceae from southern Japan with discussions on its phylogenetic relationship.

The family Thismiaceae, known as "fairy lanterns" for their urn- or bell-shaped flowers with basally fused tepals, consists of non-photosynthetic flowering monocots mainly in tropical regions, extending into subtropical and temperate areas. Here, we propose a new mycoheterotrophic genus, Relictithismia Suetsugu & Tagane (Thismiaceae), with its monotypic species Relictithismia kimotsukiensis Suetsugu, Yas.Nakam. & Tagane from Kimotsuki Mountains in the Osumi Peninsula, Kagoshima Prefecture, Kyushu Island, southern Japan. Relictithismia resembles Haplothismia Airy Shaw in having a cluster of tuberous roots, a feature previously observed only in this genus within the family Thismiaceae. However, it differs in having solitary flowers (vs. 2-6-flowered pseudo-raceme in Haplothismia), anther thecae largely separated (vs. connate), and the presence of an annulus (vs. absent). Additionally, Relictithismia differs from the geographically overlapping genus Thismia Griff. in its stamen structure and the position of the annulus. In Relictithismia, the stamens lack connectives, and its free filaments arise from the annulus located inside the perianth mouth, while in Thismia, the stamens typically have connate connectives, forming a staminal tube pendulous from the annulus located at the mouth of the floral tube. Our morphological and phylogenetic data indicated that R. kimotsukiensis holds an early-diverging position within the family, situated outside the Old World Thismia clade. This paper offers an extensive description and color photographs of R. kimotsukiensis, complemented by notes on its phylogenetic relationship and evolutionary history.

A model for the reproduction of amylopectin cluster by coordinated actions of starch branching enzyme isoforms.

Amylopectin is a highly branched glucan which accounts for approximately 65-85% of starch in most plant tissues. It is crucially important to understand the biosynthetic process of this glucan in regulating the structure and functional properties of starch granules. Currently, the most accepted ideas of structural feature and biosynthesis of amylopectin are that amylopectin is composed of a branched element called "cluster" and that the essential process of amylopectin biosynthesis is to reproduce a new cluster from the existing cluster. The present paper proposes a model explaining the whole process of amylopectin biosynthesis as to how the new cluster is reproduced by concerted actions of multiple isoforms of starch biosynthetic enzymes, particularly by combinations of distinct roles of starch branching enzyme (BE) isoforms. This model proposes for the first time the molecular mechanism as to how the formation of a new cluster is initiated, and the reason why BEI can play a major role in this step. This is because BEI has a rather broad chain-length preference compared to BEIIb, because a low preference of BEI for the substrate chain-length is advantageous for branching a couple of elongated chains that are not synchronously formed and thus these chains having varied lengths could be safely attacked by this isoform. On the contrary, it is unlikely that BEIIb is involved in this reaction because it can react to only short chains having degree of polymerization of 12-14. BEIIa is possibly able to complement the role of BEI to some extent, because BEIIa can attack basically short chains but its chain-length preference is lower compared with BEIIb. The model implies that the first branches mainly formed by BEI to construct the amorphous lamellae whereas the second branches predominantly formed by BEIIb are located mainly in the crystalline lamellae. This paper provides new insights into the roles of BEI, BEIIb, and BEIIa in amylopectin biosynthesis in cereal endosperm.

On the cluster structure of amylopectin.

KEY MESSAGE: Two opposing models for the amylopectin structure are historically and comprehensively reviewed, which leads us to a better understanding of the specific fine structure of amylopectin. Amylopectin is a highly branched glucan which accounts for approximately 65-85 of starch in most plant tissues. However, its fine structure is still not fully understood due to the limitations of current methodologies. Since the 1940 s, many scientists have attempted to elucidate the distinct structure of amylopectin. One of the most accepted concepts is that amylopectin has a structural element known as "cluster", in which neighboring side chains with a degree of polymerization of ≥ 10 in the region of their non-branched segments form double helices. The double helical structures are arranged in inter- and intra-clusters and are the origin of the distinct physicochemical and crystalline properties of starch granules. Several models of the cluster structure have been proposed by starch scientists worldwide during the progress of analytical methods, whereas no direct evidence so far has been provided. Recently, Bertoft and colleagues proposed a new model designated as "the building block and backbone (BB) model". The BB model sharply contrasts with the cluster model in that the structural element for the BB model is the building block, and that long chains are separately synthesized and positioned from short chains constituting the building block. In the present paper, we conduct the historical review of the cluster concept detailing how and when the concept was established based on experimental results by many scientists. Then, differences between the two opposing concepts are explained and both models are critically discussed, particularly from the point of view of the biochemical regulation of amylopectin biosynthesis.

Suppressed expression of starch branching enzyme 1 and 2 increases resistant starch and amylose content and modifies amylopectin structure in cassava.

KEY MESSAGE: Suppression of starch branching enzymes 1 and 2 in cassava leads to increased resistant starch content through the production of high-amylose and modification of the amylopectin structure. Cassava (Manihot esculenta Crantz) is a starchy root crop used for human consumption as a staple food and industrial applications. Starch is synthesized by various isoforms of several enzymes. However, the function of starch branching enzymes (SBEs) in starch biosynthesis and mechanisms of starch regulation in cassava have not been understood well. In this study, we aimed to suppress the expression of SBEs in cassava to generate starches with a range of distinct properties, in addition to verifying the functional characteristics of the SBEs. One SBE1, two SBE2, and one SBE3 genes were classified by phylogenetic analysis and amino acid alignment. Quantitative real-time RT-PCR revealed tissue-specific expression of SBE genes in the tuberous roots and leaves of cassava. We introduced RNAi constructs containing fragments of SBE1, SBE2, or both genes into cassava by Agrobacterium-mediated transformation, and assessed enzymatic activity of SBE using tuberous roots and leaves from these transgenic plants. Simultaneous suppression of SBE1 and SBE2 rendered an extreme starch phenotype compared to suppression of SBE2 alone. Degree of polymerization of 6-13 chains in amylopectin was markedly reduced by suppression of both SBE1 and SBE2 in comparison to the SBE2 suppression; however, no change in chain-length profiles was observed in the SBE1 suppression alone. The role of SBE1 and SBE2 may have functional overlap in the storage tissue of cassava. Simultaneous suppression of SBE1 and SBE2 resulted in highly resistant starch with increased apparent amylose content compared to suppression of SBE2 alone. This study provides valuable information for understanding starch biosynthesis and suggests targets for altering starch quality.

Changes in fine structure of amylopectin and internal structures of starch granules in developing endosperms and culms caused by starch branching enzyme mutations of japonica rice.

KEY MESSAGE: BEIIb plays a specific role in determining the structure of amylopectin in rice endosperm, whereas BEIIa plays the similar role in the culm where BEIIb is absent. Cereals have three types of starch branching enzymes (BEs), BEI, BEIIa, and BEIIb. It is widely known that BEIIb is specifically expressed in the endosperm and plays a distinct role in the structure of amylopectin because in its absence the amylopectin type changes to the amylose-extender-type (ae-type) or B-type from the wild-type or A-type and this causes the starch crystalline allomorph to the B-type from the wild-type A-type. This study aimed to clarify the role of BEIIa in the culm where BEIIb is not expressed, by using a be2a mutant in comparison with results with be2b and be1 mutants. The results showed that the amylopectin structure exhibited the B-type in the be2a culm compared with the A-type in the wild-type culm. The starch granules from the be2a culm also showed the B-type like allomorph when examined by X-ray diffraction analysis and optical sum frequency generation spectroscopy. Both amylopectin chain-length profile and starch crystalline properties were found to be the A-type at the very early stage of endosperm development at 4-6 days after pollination (DAP) even in the be2b mutant. All these results support a view that in the culm as well as in the endosperm at 4-6 DAP, BEIIa can play the role of BEIIb which has been well documented in maturing endosperm. The possible mechanism as to how BEIIa can play its role is discussed.

Estimating radiation exposure of the brain of a physician with a protective flap in interventional radiology: A phantom study.

PURPOSE: The efficiency of protective equipment for the brain has not been verified at the left anterior oblique (LAO) position, which is commonly used in clinical procedures. The purpose of this study was to investigate radiation exposure of the brain in interventional radiology (IR) and the shielding ability of a new protective flap. METHODS: We made a flap that combined a protective cap with a left lateral face shield. The flap was made of tungsten-containing rubber (TCR). An anthropomorphic head phantom was placed at the physician's position, and air kerma rates (µGy/min and µGy/15s) were measured by electronic dosimeter at three locations: the surface of the left side of the head, and the left and right temporal lobes with the protective cap and the flap in fluoroscopy and cine modes. The X-ray tube was at the lower left side of the physician, and its angles were LAO60 and LAO60CAU40. The tube voltage (95-125 kV), tube current (4.7-732 mA), and air kerma rate (27.8-1078 mGy/min) were automatically adjusted by the X-ray system. We obtained the cap and the flap shielding efficiencies. RESULTS: In cine mode at LAO60CAU40, the shielding efficiencies on the surface of the left side of the head and left temporal lobe with the cap were 92.6% and 5.1%, respectively, and the corresponding shielding efficiencies with the flap were 92.5% and 86.1%, respectively. The flap can reduce radiation exposure of the brain more than the cap alone. CONCLUSIONS: At the left anterior oblique in interventional radiology, the flap can reduce exposure to the brain.

Effects of a single dose of tablets containing lactononadecapeptide on cognitive function in healthy adults: a randomized, double-blind, cross-over, placebo-controlled trial.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE) is a memory-improving peptide. The current study aimed to determine the effects of a single dose of tablets containing LNDP on cognitive function in healthy Japanese men aged 30-59 years. A randomized, double-blind, cross-over, placebo-controlled trial was conducted in participants randomly assigned to receive LNDP or placebo tablets. The Uchida-Kraepelin test was used to induce cognitive load in participants as a model of work load. Cognitive function was evaluated using the Japanese version of the CNS Vital Signs. Composite memory and verbal memory were significantly higher following consumption of LNDP than placebo tablets. Carryover effects were observed in attention and concentration domains so that period 1 data was analyzed. LNDP consumption led to higher processing speed, executive function, and cognitive flexibility than placebo. Thus, supplementation with a single dose of LNDP tablets may improve cognitive functions including memory, attention, concentration, and information processing in daily life.

An updating approach for knowledge-based planning models to improve plan quality and variability in volumetric-modulated arc therapy for prostate cancer.

PURPOSE: The purpose of this study was to compare the dose-volume parameters and regression scatter plots of the iteratively improved RapidPlan (RP) models, specific knowledge-based planning (KBP) models, in volumetric-modulated arc therapy (VMAT) for prostate cancer over three periods. METHODS: A RP1 model was created from 47 clinical intensity-modulated radiation therapy (IMRT)/VMAT plans. A RP2 model was created to exceed dosimetric goals which set as the mean values +1SD of the dose-volume parameters of RP1 (50 consecutive new clinical VMAT plans). A RP3 model was created with more strict dose constraints for organs at risks (OARs) than RP1 and RP2 models (50 consecutive anew clinical VMAT plans). Each RP model was validated against 30 validation plans (RP1, RP2, and RP3) that were not used for model configuration, and the dose-volume parameters were compared. The Cook's distances of regression scatterplots of each model were also evaluated. RESULTS: Significant differences (p < 0.05) between RP1 and RP2 were found in Dmean (101.5% vs. 101.9%), homogeneity index (3.90 vs. 4.44), 95% isodose conformity index (1.22 vs. 1.20) for the target, V40Gy (47.3% vs. 45.7%), V60Gy (27.9% vs. 27.1%), V70Gy (16.4% vs. 15.2%), and V78Gy (0.4% vs. 0.2%) for the rectal wall, and V40Gy (43.8% vs. 41.8%) and V70Gy (21.3% vs. 20.5%) for the bladder wall, whereas only V70Gy (15.2% vs. 15.8%) of the rectal wall differed significantly between RP2 and RP3. The proportions of cases with a Cook's distance of <1.0 (RP1, RP2, and RP3 models) were 55%, 78%, and 84% for the rectal wall, and 77%, 68%, and 76% for the bladder wall, respectively. CONCLUSIONS: The iteratively improved RP models, reflecting the clear dosimetric goals based on the RP feedback (dose-volume parameters) and more strict dose constraints for the OARs, generated superior dose-volume parameters and the regression scatterplots in the model converged. This approach could be used to standardize the inverse planning strategies.

Analysis of malto-oligosaccharides and related metabolites in rice endosperm during development.

MAIN CONCLUSION: Linear glucans with degree of polymerization of up to 23 were detected in rice endosperm at the very early developmental stage of endosperm and considered to play an important role in the de novo synthesis of branched glucans. Little is known concerning the contribution of malto-oligosaccharides (MOS) and longer linear glucans to the starch biosynthesis in cereal endosperm. In the present study, the changes in the amount of major metabolic intermediates including MOS and linear glucans with a degree of polymerization (DP) of ≤ 9 and ≥ 10, respectively, in rice endosperm were measured during the development. Significant amounts of linear glucans of at least DP23 were present in the endosperm at 3 and 5 days after pollination (DAP), whereas most MOS of DP up to 8 were detected in the endosperm throughout the development up to 20 DAP. It was also found that a significant amount of simple sugars such as sucrose, glucose, and fructose, and organic acids such as malic acid, citric acid, and succinic acid were present in the developing endosperm. Although the levels of metabolites are not directly related to the extent of the metabolic flux, the present results suggest that MOS and linear glucans as well as these sugars and organic acids are involved in starch biosynthesis of rice endosperm. It is thought that linear glucans might play a role in starch biosynthesis in rice endosperm, presumably as the precursor for the subsequent synthesis of branched glucans involved in the initiation process that is possibly active in the endosperm at the very early developmental stage.

Dynamic whole-body 18F-FDG PET for differentiating abnormal lesions from physiological uptake.

PURPOSE: Serial assessment of visual change in 18F-FDG uptake on whole-body 18F-FDG PET imaging was performed to differentiate pathological uptake from physiological uptake in the urinary and gastrointestinal tracts. METHODS: In 88 suspected cancer patients, serial 3-min dynamic whole-body PET imaging was performed four times, from 60 min after 18F-FDG administration. In dynamic image evaluation, high 18F-FDG uptake was evaluated by two nuclear medicine physicians and classified as "changed" or "unchanged" based on change in uptake shape over time. Detectability of pathological uptake based on these criteria was assessed and compared with conventional image evaluation. RESULTS: Dynamic whole-body PET imaging provided images of adequate quality for visual assessment. Dynamic image evaluation was "changed" in 118/154 regions of high physiological 18F-FDG uptake (77%): in 9/19 areas in the stomach (47%), in 32/39 in the small intestine (82%), in 17/33 in the colon (52%), and in 60/63 in the urinary tract (95%). In the 86 benign or malignant lesions, 84 lesions (98%) were "unchanged." A high 18F-FDG uptake area that shows no change over time using these criteria is highly likely to represent pathological uptake, with sensitivity of 97%, specificity of 76%, PPV of 70%, NPV of 98%, and accuracy of 84%. CONCLUSION: Dynamic whole-body 18F-FDG PET imaging enabled differentiation of pathological uptake from physiological uptake in the urinary and gastrointestinal tracts, based on visual change of uptake shape.

Yeast mannan increases Bacteroides thetaiotaomicron abundance and suppresses putrefactive compound production in in vitro fecal microbiota fermentation.

Yeast mannan is a part of yeast cell wall and can potentially affect gut microflora as a soluble dietary fiber. We demonstrated that yeast mannan suppressed putrefactive production and increased the relative abundance of Bacteroides thetaiotaomicron in in vitro fecal fermentation. These results suggest that yeast mannan can be used as a novel prebiotic food ingredient.

[Usefulness of the Principle of Selective Excitation Technique Non-electrocardiogramgated 3D-TOF Method in Lower-extremity MRA Using a Whole-body Coil].

PURPOSE: Generally, non-contrast, lower-extremity, magnetic resonance angiography (MRA) uses electrocardiography; thus, examination is difficult in patients who exhibit arrhythmia. However, the use of electrocardiography can be avoided by using the selective excitation technique in non-electrocardiogram-gated three-dimensional time-of-flight (TOF) (pNon-Gate) MRA. Therefore, patients who exhibit arrhythmia can be examined with pNon-Gate MRA. The purpose of this study was to assess the usefulness of pNon-Gate MRA by using a whole-body coil. METHODS: Images acquired using pNon-Gate MRA were compared with images acquired using electrocardiogram-gated two-dimensional TOF (Gate) MRA. Comparisons comprised visual evaluation, as well as comparisons of contrast-to-noise ratio and imaging time. RESULTS: In visual evaluation of the pelvic region, Gate MRA showed better results than pNon-Gate MRA. In terms of contrast-to-noise ratio, there were no significant differences in either region. Imaging time showed no significant differences between the two methods. However, it exceeded 30 minutes frequently for Gate MRA, but rarely for pNon-Gate MRA. CONCLUSIONS: pNon-Gate MRA is useful in patients with frequent arrhythmia and in those with difficulty in maintaining long-term postures. By using a whole-body coil, pNon-Gate MRA enables easier patient positioning and setting. Therefore, pNon-Gate MRA can be used in routine examinations.

Differences in specificity and compensatory functions among three major starch synthases determine the structure of amylopectin in rice endosperm.

The lengths of amylopectin-branched chains are precise and influence the physicochemical properties of starch, which determine starch functionality. Three major isozymes of starch synthases (SSs), SSI, SSII(a), and SSIII(a), are primarily responsible for amylopectin chain elongation in the storage tissues of plants. To date, the majority of reported rice mutants were generated using japonica cultivars, which have almost inactive SSIIa. Although three SSs share some overlapping chain length preferences, whether they complement each other remains unknown due to the absence of suitable genetic combinations of materials. In this study, rice ss1/SS2a/SS3a and SS1/SS2a/ss3a were newly generated, and the chain length distribution patterns of all the possible combinations of presence and absence of SSI, SSIIa, and SSIIIa activities were compared. This study demonstrated that SSIIa can complement most SSI functions that use glucan chains with DP 6-7 to generate DP 8-12 chains but cannot fully compensate for the elongation of DP 16-19 chains. This suggests that SSIIa preferentially elongates outer but not inner chains of amylopectin. In addition, the results revealed that neither SSI nor SSIIIa compensate for SSIIa. Neither SSI nor SSIIa compensate for elongation of DP >30 by SSIIIa. SSIIa could not resolve the pleiotropic increase of SSI caused by the absence of SSIIIa; instead, SSIIa further elongated those branches elongated by SSI. These results revealed compensatory differences among three major SS isozymes responsible for lengths of amylopectin branches.

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm.

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.

Characterization of Function of the GlgA2 Glycogen/Starch Synthase in Cyanobacterium sp. Clg1 Highlights Convergent Evolution of Glycogen Metabolism into Starch Granule Aggregation.

At variance with the starch-accumulating plants and most of the glycogen-accumulating cyanobacteria, Cyanobacterium sp. CLg1 synthesizes both glycogen and starch. We now report the selection of a starchless mutant of this cyanobacterium that retains wild-type amounts of glycogen. Unlike other mutants of this type found in plants and cyanobacteria, this mutant proved to be selectively defective for one of the two types of glycogen/starch synthase: GlgA2. This enzyme is phylogenetically related to the previously reported SSIII/SSIV starch synthase that is thought to be involved in starch granule seeding in plants. This suggests that, in addition to the selective polysaccharide debranching demonstrated to be responsible for starch rather than glycogen synthesis, the nature and properties of the elongation enzyme define a novel determinant of starch versus glycogen accumulation. We show that the phylogenies of GlgA2 and of 16S ribosomal RNA display significant congruence. This suggests that this enzyme evolved together with cyanobacteria when they diversified over 2 billion years ago. However, cyanobacteria can be ruled out as direct progenitors of the SSIII/SSIV ancestral gene found in Archaeplastida. Hence, both cyanobacteria and plants recruited similar enzymes independently to perform analogous tasks, further emphasizing the importance of convergent evolution in the appearance of starch from a preexisting glycogen metabolism network.

Functional characterization of three (GH13) branching enzymes involved in cyanobacterial starch biosynthesis from Cyanobacterium sp. NBRC 102756.

Starch and glycogen are widespread storage polysaccharides in bacteria, plants, and animals. Recently, some cyanobacteria were found to accumulate water-insoluble α-glucan similar to amylopectin rather than glycogen, the latter of which is more commonly produced in these organisms. The amylopectin-producing species including Cyanobacterium sp. NBRC 102756 invariably have three branching enzyme (BE) homologs, BE1, BE2, and BE3, all belonging to the glycoside hydrolase family 13. Multiple BE isoforms in prokaryotes have not been previously studied. In the present work, we carried out functional characterization of these enzymes expressed in Escherichia coli. The recombinant enzymes were all active, although the specific activity of BE3 was much lower than those of BE1 and BE2. After the incubation of the enzymes with amylopectin or amylose, the reaction products were analyzed by fluorophore-assisted carbohydrate capillary electrophoresis method. BE1 and BE2 showed similar chain-length preference to BEIIb isoform of rice (Oryza sativa L.), while the catalytic specificity of BE3 was similar to that of rice BEI. These results indicate that starch-producing cyanobacteria have both type-I BE (BE3) and type-II BEs (BE1 and BE2) in terms of chain-length preferences, as is the case of plants. All BE isoforms were active against phosphorylase limit dextrin, in which outer branches had been uniformly diminished to 4 glucose residues. Based on its catalytic properties, BE3 was assumed to have a role to transfer the glucan chain bearing branch(es) to give rise to a newly growing unit, or cluster as observed in amylopectin molecule.

Convergent evolution of polysaccharide debranching defines a common mechanism for starch accumulation in cyanobacteria and plants.

Starch, unlike hydrosoluble glycogen particles, aggregates into insoluble, semicrystalline granules. In photosynthetic eukaryotes, the transition to starch accumulation occurred after plastid endosymbiosis from a preexisting cytosolic host glycogen metabolism network. This involved the recruitment of a debranching enzyme of chlamydial pathogen origin. The latter is thought to be responsible for removing misplaced branches that would otherwise yield a water-soluble polysaccharide. We now report the implication of starch debranching enzyme in the aggregation of semicrystalline granules of single-cell cyanobacteria that accumulate both glycogen and starch-like polymers. We show that an enzyme of analogous nature to the plant debranching enzyme but of a different bacterial origin was recruited for the same purpose in these organisms. Remarkably, both the plant and cyanobacterial enzymes have evolved through convergent evolution, showing novel yet identical substrate specificities from a preexisting enzyme that originally displayed the much narrower substrate preferences required for glycogen catabolism.

Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.

Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch.

Lactobacillus helveticus-fermented milk improves learning and memory in mice.

OBJECTIVES: To investigate the effects of Calpis sour milk whey, a Lactobacillus helveticus-fermented milk product, on learning and memory. METHODS: We evaluated improvement in scopolamine-induced memory impairment using the spontaneous alternation behaviour test, a measure of short-term memory. We also evaluated learning and working memory in mice using the novel object recognition test, which does not involve primary reinforcement (food or electric shocks). A total of 195 male ddY mice were used in the spontaneous alternation behaviour test and 60 in the novel object recognition test. RESULTS: Forced orally administered Calpis sour milk whey powder (200 and 2000 mg/kg) significantly improved scopolamine-induced cognitive impairments (P < 0.05 and P < 0.01, respectively) and object recognition memory (2000 mg/kg; P < 0.05). DISCUSSION: These results suggest that Calpis sour milk whey may be useful for the prevention of neurodegenerative disorders, such as Alzheimer's disease, and enhancing learning and memory in healthy human subjects; however, human clinical studies are necessary.