Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.     

Involvement of cumulus cells stimulated by FSH in chromatin condensation and the activation of maturation-promoting factor in bovine oocytes

The effects of FSH-stimulated cumulus cells on the regulatory mechanisms of chromatin condensation and maturation-promoting factor (MPF) activation around the time of germinal vesicle breakdown (GVBD) in bovine oocytes were examined. Chromatin condensation occurred in oocytes arrested at the germinal vesicle (GV) stage by protein synthesis inhibitor, cycloheximide, but this condensation was blocked by FSH-stimulated cumulus cells. However, treatment with cyclic AMP (cAMP)-dependent protein kinase inhibitor, H-8, dramatically increased the proportion of oocytes possessing GVs with condensed bivalents. Under the condition of inhibited protein synthesis, the phosphorylation form of p34cdc2 kinase was not changed due to chromatin condensation, although the activity of histone H1 kinase was significantly increased compared with that of oocytes possessing GVs with filamentous bivalents. The cycloheximide-dependent GVBD block was overcome by okadaic acid (OA) in 48 and 13% of the oocytes in the absence and presence of FSH, respectively. An initial 6-h culture period critical for protein synthesis was necessary for OA to counteract the inhibitory effect exerted by cycloheximide on the induction of GVBD and activation of histone H1 kinase in the absence of FSH, whereas this first culture period was prolonged for 2 h in the presence of FSH. Furthermore, even in FSH-stimulated oocytes, H-8 facilitated an OA-counteracted overcome of the cycloheximide-dependent GVBD block after 2 h of initial culture for protein synthesis. From these results, it is concluded that cAMP-dependent protein kinase activity regulated by cumulus cells following FSH-stimulation requests plays a role in the complex mechanism of chromatin condensation and MPF activation leading to meiotic resumption in bovine oocytes.     

A Novel Phthalimide Derivative,TC11, Has Preclinical Effects on High-Risk Myeloma Cells and Osteoclasts

Despite the recent advances in the treatment of multiple myeloma (MM), MM patients with high-risk cytogenetic changes such as t(4;14) translocation or deletion of chromosome 17 still have extremely poor prognoses. With the goal of helping these high-risk MM patients, we previously developed a novel phthalimide derivative, TC11. Here we report the further characterization of TC11 including anti-myeloma effects in vitro and in vivo, a pharmacokinetic study in mice, and anti-osteoclastogenic activity. Intraperitoneal injections of TC11 significantly delayed the growth of subcutaneous tumors in human myeloma-bearing SCID mice. Immunohistochemical analyses showed that TC11 induced apoptosis of MM cells in vivo. In the pharmacokinetic analyses, the C_max was 2.1 μM at 1 h after the injection of TC11, with 1.2 h as the half-life. TC11 significantly inhibited the differentiation and function of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts in mouse osteoclast cultures using M-CSF and RANKL. We also revealed that TC11 induced the apoptosis of myeloma cells accompanied by α-tubulin fragmentation. In addition, TC11 and lenalidomide, another phthalimide derivative, directly bound to nucleophosmin 1 (NPM1), whose role in MM is unknown. Thus, through multiple molecular interactions, TC11 is a potentially effective drug for high-risk MM patients with bone lesions. The present results suggest the possibility of the further development of novel thalidomide derivatives by drug designing.     

Temporal and Motor Representation of Rhythm in Fronto-Parietal Cortical Areas: An fMRI Study

When sounds occur with temporally structured patterns, we can feel a rhythm. To memorize a rhythm, perception of its temporal patterns and organization of them into a hierarchically structured sequence are necessary. On the other hand, rhythm perception can often cause unintentional body movements. Thus, we hypothesized that rhythm information can be manifested in two different ways; temporal and motor representations. The motor representation depends on effectors, such as the finger or foot, whereas the temporal representation is effector-independent. We tested our hypothesis with a working memory paradigm to elucidate neuronal correlates of temporal or motor representation of rhythm and to reveal the neural networks associated with these representations. We measured brain activity by fMRI while participants memorized rhythms and reproduced them by tapping with the right finger, left finger, or foot, or by articulation. The right inferior frontal gyrus and the inferior parietal lobule exhibited significant effector-independent activations during encoding and retrieval of rhythm information, whereas the left inferior parietal lobule and supplementary motor area (SMA) showed effector-dependent activations during retrieval. These results suggest that temporal sequences of rhythm are probably represented in the right fronto-parietal network, whereas motor sequences of rhythm can be represented in the SMA-parietal network.     

The Relationship between Processing Speed and Regional White Matter Volume in Healthy Young People

Processing speed is considered a key cognitive resource and it has a crucial role in all types of cognitive performance. Some researchers have hypothesised the importance of white matter integrity in the brain for processing speed; however, the relationship at the whole-brain level between white matter volume (WMV) and processing speed relevant to the modality or problem used in the task has never been clearly evaluated in healthy people. In this study, we used various tests of processing speed and Voxel-Based Morphometry (VBM) analyses, it is involves a voxel-wise comparison of the local volume of gray and white, to assess the relationship between processing speed and regional WMV (rWMV). We examined the association between processing speed and WMV in 887 healthy young adults (504 men and 383 women; mean age, 20.7 years, SD, 1.85). We performed three different multiple regression analyses: we evaluated rWMV associated with individual differences in the simple processing speed task, word–colour and colour–word tasks (processing speed tasks with words) and the simple arithmetic task, after adjusting for age and sex. The results showed a positive relationship at the whole-brain level between rWMV and processing speed performance. In contrast, the processing speed performance did not correlate with rWMV in any of the regions examined. Our results support the idea that WMV is associated globally with processing speed performance regardless of the type of processing speed task.     

Effect of apple intake on fecal microbiota and metabolites in humans

The effects of apple intake on the fecal flora, water content, pH, and metabolic activities in eight healthy volunteers and the utilization of apple pectin in vitro were investigated. Although several isolates of Bifidobacterium, Lactobacillus, Enterococcus, and the Bacteroides fragilis group utilized apple pectin, most isolates of Escherichia coli, Collinsela aerofaciense, Eubacterium limosum, and Clostridium perfringens could not. When fecal samples from healthy adults were incubated in liquid broth with apple pectin present or absent, the numbers of Bifidobacterium and Lactobacillus in the former were higher than those in the later. After the intake of apples (2 apples a day for 2 weeks) by eight healthy adult humans, the number of bifidobacteria in feces increased (p < 0.05 on day 7 and p < 0.01 on day 14 of the intake period), and the numbers of Lactobacillus and Streptococcus including Enterococcus tended to increase. However, lecithinase-positive clostridia, including C. perfringens, decreased (p < 0.05), and Enterobacteriaceae and Pseudomonas tended to decrease. Moreover, the concentrations of fecal acetic acid tended to increase on apple intake. The fecal ammonia concentration showed a tendency to reduce and fecal sulfide decreased (p < 0.05) on apple intake. These findings indicate that apple consumption is related to an improved intestinal environment, and apple pectin is one of the effective apple components improving the fecal environment.     

Development of an efficient gene targeting system in Colletotrichum higginsianum using a non-homologous end-joining mutant and Agrobacterium tumefaciens-mediated gene transfer

The hemibiotrophic ascomycete Colletotrichum higginsianum is the casual agent of anthracnose disease of cruciferous plants. High efficiency transformation by Agrobacterium tumefaciens-mediated gene transfer has been established for this fungus. However, targeted gene mutagenesis through homologous recombination rarely occurs in C. higginsianum. We have identified and disrupted the C. higginsianum homologue of the human Ku70 gene, ChKU70, which encodes a protein that plays a role in non-homologous end-joining for repair of DNA breaks. chku70 mutants showed a dramatic increase in the frequency of integration of introduced exogenous DNA fragments by homologous recombination without any detectable phenotypic defects. This result demonstrates that the chku70 mutant is an efficient recipient for targeted gene mutagenesis in C. higginsianum. We have also developed a novel approach [named direct repeat recombination-mediated gene targeting (DRGT)] for targeted gene disruption through Agrobacterium tumefaciens-mediated gene transfer. DRGT utilizes homologous recombination between repeated sequences on the T-DNA flanking a partial fragment of the target gene. Our results suggest that DRGT in the chku70 mutant background could be a useful tool for rapid isolation of targeted gene disruptants in C. higginsianum.     

Aggregate Size and Architecture Determine Microbial Activity Balance for One-Stage Partial Nitritation and Anammox

Aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB) cooperate in partial nitritation/anammox systems to remove ammonium from wastewater. In this process, large granular microbial aggregates enhance the performance, but little is known about granulation so far. In this study, three suspended-growth oxygen-limited autotrophic nitrification-denitrification (OLAND) reactors with different inoculation and operation (mixing and aeration) conditions, designated reactors A, B, and C, were used. The test objectives were (i) to quantify the AerAOB and AnAOB abundance and the activity balance for the different aggregate sizes and (ii) to relate aggregate morphology, size distribution, and architecture putatively to the inoculation and operation of the three reactors. A nitrite accumulation rate ratio (NARR) was defined as the net aerobic nitrite production rate divided by the anoxic nitrite consumption rate. The smallest reactor A, B, and C aggregates were nitrite sources (NARR, >1.7). Large reactor A and C aggregates were granules capable of autonomous nitrogen removal (NARR, 0.6 to 1.1) with internal AnAOB zones surrounded by an AerAOB rim. Around 50% of the autotrophic space in these granules consisted of AerAOB- and AnAOB-specific extracellular polymeric substances. Large reactor B aggregates were thin film-like nitrite sinks (NARR, <0.5) in which AnAOB were not shielded by an AerAOB layer. Voids and channels occupied 13 to 17% of the anoxic zone of AnAOB-rich aggregates (reactors B and C). The hypothesized granulation pathways include granule replication by division and budding and are driven by growth and/or decay based on species-specific physiology and by hydrodynamic shear and mixing.In the last few years, autotrophic nitrogen removal via partial nitritation and anoxic ammonium oxidation (anammox) has evolved from lab- to full-scale treatment of nitrogenous wastewaters with a low biodegradable organic compound content, and this evolution has been driven mainly by a significant decrease in the operational costs compared to the costs of conventional nitrification and heterotrophic denitrification (11, 23). Oxygen-limited autotrophic nitrification and denitrification (OLAND) is one of the autotrophic processes used and is a one-stage procedure; i.e., partial nitritation and anammox occur in the same reactor (30). The “functional” autotrophic microorganisms in OLAND include aerobic ammonium-oxidizing bacteria (AerAOB) and anoxic ammonium-oxidizing bacteria (AnAOB). With oxygen, AerAOB oxidize ammonium to nitrite (nitritation), and with the nitrite AnAOB oxidize the residual ammonium to form dinitrogen gas and some nitrate (anammox). Additional aerobic nitrite oxidation to nitrate (nitratation) by nitrite-oxidizing bacteria (NOB) lowers the nitrogen removal efficiency, but it can, for instance, be prevented at low dissolved oxygen (DO) levels because the oxygen affinity of AerAOB is higher than that of NOB (16). Reactor configurations for the OLAND process can be based on suspended biomass growing in aggregates, like that in a sequencing batch reactor (SBR) (37) or a gas lift or upflow reactor (32). For suspended-growth systems there are two important challenges: biomass retention and equilibrated microbial activities.High biomass retention efficiency is a prerequisite in anammox technologies because of the slow growth of AnAOB (33). In suspended biomass systems, settling properties determine the retention of biomass and are related to the microbial aggregate morphology (floc or granule) and size. Granules can be defined as compact and dense aggregates with an approximately spherical external appearance that do not coagulate under decreased hydrodynamic shear conditions and settle significantly faster than flocs (18). Toh and coworkers calculated a lower sludge volume index for aerobic granules than for aerobic flocs and also showed that there was an increase in the settling velocity with increasing granule size (35). Hence, in terms of physical properties, large granules are preferable for suspended-growth applications.OLAND aggregate size not only influences settling properties but also affects the proportion of microbial nitrite production and consumption; lower AerAOB activity and higher AnAOB activity were observed with larger aggregates (25, 37). Theoretically, a microbial aggregate with equal nitrite production and nitrite consumption can remove ammonium autonomously, because of its independence from other aggregates for acquisition and conversion of nitrite. Hence, with an increasing aggregate size and thus with a decreasing ratio of nitrite production to nitrite consumption, three functional categories of aggregates can be distinguished: nitrite sources, autonomous nitrogen removers, and nitrite sinks. Because minimal nitrite accumulation is one of the prerequisites for high nitrogen removal efficiency in OLAND reactors, the presence of excess small aggregates is undesirable (9, 37).Although large granular aggregates are desirable for biomass retention and activity balance, so far no formation mechanisms have been proposed for OLAND granules, in contrast to the well-studied anaerobic (13) and aerobic (1) granules. In order to determine general and environment-specific determinants for aggregate size and architecture, three suspended-growth OLAND reactors with different inoculation and operation (mixing and aeration) parameters were selected, and these reactors were designated reactors A, B, and C (Table ​(Table1).1). The first objective of this study was to gain more insight into the relationship between OLAND aggregate size, AerAOB and AnAOB abundance, and the activity balance. The second objective was to propose pathways for aggregation and granulation by relating (dis)similarities in aggregate size distribution, morphology, and architecture to differences in reactor inoculation and operation.

TABLE 1.

Overview of the three OLAND reactor systems from which suspended biomass samples were obtained