AUF1, an mRNA decay factor, has a discordant role in Cpeb1 expression.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates polyadenylation and subsequent translation of CPE-containing mRNAs involved in various physiological and pathological phenomena. Although the significance of CPEB1-mediated translational regulation has recently been reported, the detailed regulatory mechanism of Cpeb1 expression remains unclear. To elucidate the post-transcriptional regulatory mechanisms of Cpeb1 expression, we constructed reporter plasmids containing various deletions or mutations in the Cpeb1 mRNA 3' untranslated region (3'UTR). We investigated their expression levels in Neuro2a neuroblastoma cells. We found that Cpeb1 expression is regulated through an AU-rich element in its 3'UTR. Furthermore, the mRNA decay factor AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression, and knockdown of AUF1 upregulates Cpeb1 mRNA expression but results in a decrease in CPEB1 protein levels. These findings indicate that AUF1 has a discordant role in the expression of Cpeb1.

Kinesin superfamily motor proteins and intracellular transport.

Intracellular transport is fundamental for cellular function, survival and morphogenesis. Kinesin superfamily proteins (also known as KIFs) are important molecular motors that directionally transport various cargos, including membranous organelles, protein complexes and mRNAs. The mechanisms by which different kinesins recognize and bind to specific cargos, as well as how kinesins unload cargo and determine the direction of transport, have now been identified. Furthermore, recent molecular genetic experiments have uncovered important and unexpected roles for kinesins in the regulation of such physiological processes as higher brain function, tumour suppression and developmental patterning. These findings open exciting new areas of kinesin research.

Surgical anatomy of the pelvis for total pelvic exenteration with distal sacrectomy: a cadaveric study.

PURPOSE: Intraoperative bleeding from the pelvic venous structures is one of the most serious complications of total pelvic exenteration with distal sacrectomy. The purpose of this study was to investigate the topographic anatomy of these veins and the potential source of the bleeding in cadaver dissections. METHODS: We dissected seven cadavers, focusing on the veins in the surgical resection line for total pelvic exenteration with distal sacrectomy. RESULTS: The presacral venous plexus and the dorsal vein complex are thin-walled, plexiform, and situated on the line of resection. The internal iliac vein receives blood from the pelvic viscera and the perineal and the gluteal regions and then crosses the line of resection as a high-flow venous system. It has abundant communications with the presacral venous plexus and the dorsal vein complex. CONCLUSION: The anatomical features of the presacral venous plexus, the dorsal vein complex, and the internal iliac vein make them highly potential sources of bleeding. Surgical management strategies must consider the anatomy and hemodynamics of these veins carefully to perform this procedure safely.

Couinaud Type A communicating accessory bile duct: report of a case.

BACKGROUND: Communicating accessory bile ducts are defined as ducts that communicate between major biliary channels but do not drain individual segments of the liver. The Couinaud Type A communicating accessory bile duct is a rare anomaly where an aberrant duct connects the right main hepatic duct to the common hepatic duct without segmental drainage. There are very few reports of this anomaly in the literature to date. CASE PRESENTATION: A 75-year-old male who died of ischemic heart disease donated his body for cadaveric dissection, which included careful attention to the anatomy of the hepatic hilum. During dissection, it was found that the right hepatic duct was duplicated and an accessory duct drained directly into the common hepatic duct. Although rare and difficult to visualize even with modern preoperative imaging techniques, sound knowledge of this rare anatomic variation is imperative to avoid inadvertent intraoperative biliary injuries which can lead to severe morbidity. CONCLUSIONS: An aberrant bile duct from the right hepatic duct to the common hepatic duct (Couinaud Type A) is an uncommon accessory bile duct that one must be aware of when performing complex hepatobiliary procedures such as right liver resection for living-related donation. Detailed preoperative imaging and careful dissection with anticipation of anomalous anatomy are of the utmost importance for the safe conduct of hepatic surgery.

Oxysterol-binding protein-related protein (ORP) 6 localizes to the ER and ER-plasma membrane contact sites and is involved in the turnover of PI4P in cerebellar granule neurons.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved lipid binding proteins found in organisms ranging from yeast to mammals. Recent findings have indicated that these proteins mainly localize to contact sites of 2 different membranous organelles. ORP6, a member of the ORP subfamily III, is one of the least studied ORPs. Using approaches in molecular cell biology, we attempted to study the characteristics of ORP6 and found that ORP6 is abundantly expressed in mouse cultured neurons. Deconvolution microscopy of cultured cerebellar granular cells revealed that ORP6 is localized to the endoplasmic reticulum (ER) and ER-plasma membrane (PM) contact sites, where it co-localized with extended synaptotagmin2 (E-Syt2), a well-known ER-PM contact site marker. E-Syt2 also co-localized with ORP3, another subfamily III member, and ORP5, a subfamily IV member. However, ORP5 does not distribute to the same ER-PM contact sites as subfamily III members. Also, the co-expression of ORP3 but not ORP5 altered the distribution of ORP6 into the processes of cerebellar neurons. Immunoprecipitation demonstrated binding between the intermediate region of ORP6 and ORP3 or ORP6 itself. Additionally, the localization of ORP6 in the PM decreased when co-expressed with the intermediate region of ORP6, in which the pleckstrin homology (PH) domain and OSBP-related ligand binding domain (ORD) are deleted. Over-expression of this intermediate region shifted the location of a phophtidylinositol-4-phosphate (PI4P) marker from the Golgi to the PM. Knockdown of ORP6 resulted in the same shift of the PI4P marker. Collectively, our data suggests that the recruitment of ORP6 to ER-PM contact sites is involved in the turnover of PI4P in cerebellar granular neurons.

Reappraisal of the lateral rectal ligament: an anatomical study of total mesorectal excision with autonomic nerve preservation.

PURPOSE: The term "lateral rectal ligament" in surgery for rectal cancer has caused confusion regarding its true existence and contents. In previous studies, investigators claimed the existence of the ligament and described its topographical features as neurovascular structures and their surrounding connective tissues located at the anterolateral aspect of the distal rectum or the posterolateral aspect of the middle rectum. The purpose of this study is to evaluate the structure of the so-called "lateral rectal ligament" in cadaver dissections. METHODS: Dissection was performed in nine cadavers (eight males and one female, aged 73 to 94 years) in accordance with typical total mesorectal excision techniques. During dissection, structures related to "the ligament" were examined and images recorded. RESULTS: At the anterolateral aspect of the distal rectum, the middle rectal artery was noted to be crossing the fusion of Denonvilliers' fascia and the proper rectal fascia. At the posterolateral aspect of the middle rectum, there was a structure which consisted of the rectal nerves running through the fusion of the pelvic fasciae. Although called "ligaments," neither structure contained discrete strong connective tissue fixing the rectum to the pelvic wall. CONCLUSIONS: The proper rectal fascia and surrounding pelvic fasciae fuse firmly anterolaterally and posterolaterally where neurovascular structures course toward the rectum. During a total mesorectal excision, the surgical dissection plane coincides with the fused part of the fasciae, which had long been considered the "lateral rectal ligament."

Mechanism of the Dendritic Translation and Localization of Brain-derived Neurotrophic Factor.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3' UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3' UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.

The Anticonvulsant Zonisamide Inhibits Hydroxyl Radical Generated from Methylguanidine.

Methylguanidine (MG) is a known nephrotoxin and neurotoxin, and an intracisternal injection of MG can induce convulsions in experimental animals. In this in vitro study, we examined the inhibitory effects of the antiepileptic agent zonisamide (ZNS) on hydroxyl radicals (•OH) generated from MG by using an electron spin resonance (ESR) technique. ZNS scavenged •OH generated from MG in a dose-dependent manner through direct scavenging during the auto-oxidation of MG. The rate constant of ZNS reacting with the •OH was at a near diffusion-controlled rate. These findings indicate that ZNS might detoxify MG and could thus protect against convulsive disorders.

Phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) regulates neuronal microtubule depolymerase kinesin, KIF2A and suppresses elongation of axon branches.

Neuronal morphology is regulated by cytoskeletons. Kinesin superfamily protein 2A (KIF2A) depolymerizes microtubules (MTs) at growth cones and regulates axon pathfinding. The factors controlling KIF2A in neurite development remain totally elusive. Here, using immunoprecipitation with an antibody specific to KIF2A, we identified phosphatidylinositol 4-phosphate 5-kinase alpha (PIPKα) as a candidate membrane protein that regulates the activity of KIF2A. Yeast two-hybrid and biochemical assays demonstrated direct binding between KIF2A and PIPKα. Partial colocalization of the clusters of punctate signals for these two molecules was detected by confocal microscopy and photoactivated localization microscopy. Additionally, the MT-depolymerizing activity of KIF2A was enhanced in the presence of PIPKα in vitro and in vivo. PIPKα suppressed the elongation of axon branches in a KIF2A-dependent manner, suggesting a unique PIPK-mediated mechanism controlling MT dynamics in neuronal development.

[Results of a questionnaire on efforts to increase research-oriented doctors].

We surveyed medical and dental schools to promote the exchange of information about university efforts to increase the number of research-oriented doctors. Periods in which students rotate through laboratories to conduct research were reported by more than two thirds of universities. Many comments asserted that these efforts are effective. However, a small number of respondents reported low student motivation and insufficient time for laboratory experience. MD-PhD courses, in which students take a leave of absence in the middle of undergraduate training and follow a PhD curriculum, have been employed by more than 10 universities. However, relatively few students have chosen such programs. Modified MD-PhD courses have recently been introduced by several universities. In these courses, by taking part of the graduate school curriculum in advance, undergraduate students can shorten the time they spend in graduate school. Students who take such courses are increasing. There were many opinions that extra positions and financial support for research-oriented doctors are effective and should be enhanced. There were also many opinions that emphasize the importance of identifying research-oriented students, improving laboratory working environments, attending academic meetings and inter-university consortia to maintain students' motivation, and promoting collaboration with departments of clinical medicine.

The involvement of oxysterol-binding protein related protein (ORP) 6 in the counter-transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons.

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.

Cpeb1 expression is post-transcriptionally regulated by AUF1, CPEB1, and microRNAs.

Cytoplasmic polyadenylation element binding protein 1 (CPEB1) regulates the translation of numerous mRNAs. We previously showed that AU-rich binding factor 1 (AUF1) regulates Cpeb1 expression through the 3' untranslated region (3'UTR). To investigate the molecular basis of the regulatory potential of the Cpeb1 3'UTR, here we performed reporter analyses that examined expression levels of Gfp reporter mRNA containing the Cpeb1 3'UTR. Our findings indicate that CPEB1 represses the translation of Cpeb1 mRNA and that miR-145a-5p and let-7b-5p are involved in the reduction in Cpeb1 expression in the absence of AUF1. These results suggest that Cpeb1 expression is post-transcriptionally regulated by AUF1, CPEB1, and microRNAs.

Cytoplasmic Polyadenylation Element-Binding Protein 1 Post-transcriptionally Regulates Fragile X Mental Retardation 1 Expression Through 3' Untranslated Region in Central Nervous System Neurons.

Fragile X syndrome (FXS) is an inherited intellectual disability caused by a deficiency in Fragile X mental retardation 1 (Fmr1) gene expression. Recent studies have proposed the importance of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in FXS pathology; however, the molecular interaction between Fmr1 mRNA and CPEB1 has not been fully investigated. Here, we revealed that CPEB1 co-localized and interacted with Fmr1 mRNA in hippocampal and cerebellar neurons and culture cells. Furthermore, CPEB1 knockdown upregulated Fmr1 mRNA and protein levels and caused aberrant localization of Fragile X mental retardation protein in neurons. In an FXS cell model, CPEB1 knockdown upregulated the mRNA levels of several mitochondria-related genes and rescued the intracellular heat shock protein family A member 9 distribution. These findings suggest that CPEB1 post-transcriptionally regulated Fmr1 expression through the 3' untranslated region, and that CPEB1 knockdown might affect mitochondrial function.

[Final report of the working group for the future planning of the Japanese Association of Anatomists].

The working group for the future planning of the Japanese Association of Anatomists (JAA) has been working to address the issues that were consulted from the president of JAA since October 2009. After making the interim report in March 2010, a public hearing for general members of the JAA was held and a final report was submitted to the President in January 2011. The report contains the analysis of the current situation, the directions in which we should proceed, and recommendations of concrete actions that JAA should take for each issue. The issues discussed were as follows: 1. Future prospects of anatomy and morphological sciences. How can we maintain the specialties of morphological and anatomical sciences in the rapidly advancing field of life sciences and develop collaborations with other fields? 2. Improvement of the JAA academic meetings. How can we increase JAA members and young participants in the academic meetings of the JAA? 3. Fostering the next generation of young researchers. How can we increase young researchers graduated from the schools of Medicine or Dentistry? 4. Future prospects of education of gross anatomy. Prospects of education in gross anatomy and the body donation registration system in relation with some new cadaver-related movements.

Effect of long-term confinement on metabolic and physiological parameters in mice.

Long-term and mild confinement or isolation in an enclosed environment can occur in situations such as disasters, specific political, economic or social events, nuclear shelters, seabed exploration, polar expeditions, and space travel. To investigate the effects of stress caused by long-term confinement in an enclosed environment in mammals, we divided 8-week-old C57BL/6J mice into four groups that were housed in a closed environment with a narrow metabolic cage (stress group), normal metabolic cage (control group), conventional cage (conventional group) or conventional cage with wire mesh floor (wire mesh group). The phenotypes of the mice were examined for four weeks, followed by behavioral tests. Weight gain suppression was observed in the stress group. Continuous analysis of these mice every two minutes for four weeks using an implanted measuring device showed a significantly decreased amount of spontaneous activity and subcutaneous temperature in the stress group. After housing in each environment for four weeks, the behavioral tests of mice in the stress group also revealed a shorter latency to fall off in the rotarod test and shorter stride length and interstep distance in the footprint test. Interestingly, the lower spontaneous activity of mice in the stress group was rescued by housing in conventional cages. These results suggest a temporary effect of long-term confinement in an enclosed environment as a chronic and mild stress on homeostasis in mammals.

Identification of enzymes and regulatory proteins in Escherichia coli that are oxidized under nitrogen, carbon, or phosphate starvation.

Using proteomic technologies, we identified 62 proteins that are oxidized to carbonyl derivatives during growth of Escherichia coli under nitrogen starvation (NS), carbon starvation (CS), and phosphate starvation (PS) conditions. The carbonylated proteins were converted to 2,4-dinitrophenylhydrazone derivatives and these were identified using Western blotting and mass spectrometry by searching E. coli proteins in the Swiss-Prot and/or NCBI databases. Fourteen of the oxidized proteins were formed under both NS and CS conditions, and only three proteins were specifically oxidized under PS conditions. Interestingly, the carbonyl content of proteins in crude extracts of cells harvested after 48 h of stationary growth under NS and CS was significantly lower than that observed at mid-log and end-log phases of growth. In contrast, the carbonyl content of proteins in extracts of cells grown under PS conditions was fairly constant during comparable periods of growth.

[Effect of repeated intravitreal bevacizumab injections for secondary macular edema of branch retinal vein occlusion].

PURPOSE: To evaluate the effects of intravitreal bevacizumab (IVB) injections for secondary macular edema of branch retinal vein occlusion (BRVO). SUBJECTS AND METHODS: We treated 91 patients (91 eyes) with IVB injections (1.25 mg/0.05 ml), including 27 eyes which received two injections. Visual acuity and central retinal thickness (CRT) were measured at 1, 4, 8 and 12 weeks after injection. RESULTS: The mean visual acuity and CRT improved from 0.25 (610.8 microm) at baseline to 0.47 (238.4 microm) 4 weeks after injection and 0.45 (368.7 microm) after 12 weeks. Twenty seven eyes among the total of 91 eyes had a second injection due to recurrence or worsened metamorphopsia. In these cases, mean visual acuity and CRT improved from 0.33 (483.7 microm) at baseline to 0.44 (234.3 microm) 4 weeks after injection and 0.42 (296.8 microm) after 12 weeks. Comparing the efficacy by the number of treatments, visual acuity and CRT improved more significantly in a first time treatment group. CONCLUSIONS: IVB injection is generally effective, but recurrence occurred in 26/47 eyes based on CRT. The second injection is effective, however, its effect is weak when compared with the first injection.

Role of KIFC3 motor protein in Golgi positioning and integration.

KIFC3, a microtubule (MT) minus end-directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3-/- cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3-/- cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3-/- cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3-/- cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end-directed motors other than KIFC3, such as dynein, in kifC3-/- cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3-/- cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.

Inhibitory effect of antioxidants on hydroxyl radical generation from methylguanidine: an ESR study.

Methylguanidine (MG) is known as not only a nephrotoxin but also as a neurotoxine. We have previously showed that MG itself generates hydroxyl radicals (*OH) in an in vitro study. In this study, we examined the inhibitory effects of ascorbate, EPC-K(1) (alpha-tocopheryl-L-ascorbate-2-O-phosphate diester), Trolox (water-soluble vitamin E analogue), and glutathione (GSH) on *OH generation from MG using an electron spin resonance (ESR) spectrometry with spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). It was found that these compounds have potent inhibitory effect on *OH generation from MG in the order of ascorbate > GSH > EPC-K(1) > Trolox.

Synaptic vesicle-bound pyruvate kinase can support vesicular glutamate uptake.

Glucose metabolism is essential for normal brain function and plays a vital role in synaptic transmission. Recent evidence suggests that ATP synthesized locally by glycolysis, particularly via glyceraldehyde 3-phosphate dehydrogenase/3-phosphoglycerate kinase, is critical for synaptic transmission. We present evidence that ATP generated by synaptic vesicle-associated pyruvate kinase is harnessed to transport glutamate into synaptic vesicles. Isolated synaptic vesicles incorporated [(3)H]glutamate in the presence of phosphoenolpyruvate (PEP) and ADP. Pyruvate kinase activators and inhibitors stimulated and reduced PEP/ADP-dependent glutamate uptake, respectively. Membrane potential was also formed in the presence of pyruvate kinase activators. "ATP-trapping" experiments using hexokinase and glucose suggest that ATP produced by vesicle-associated pyruvate kinase is more readily used than exogenously added ATP. Other neurotransmitters such as GABA, dopamine, and serotonin were also taken up into crude synaptic vesicles in a PEP/ADP-dependent manner. The possibility that ATP locally generated by glycolysis supports vesicular accumulation of neurotransmitters is discussed.