山田 晃司

INTRODUCTION: The decline in spatial working memory is one of the earliest signs of normal brain aging. OBJECTIVE: We developed a novel physical exercise method, termed the "shaking exercise," to slow down this process. METHODS: The experimental protocol included administering the shaking exercise for 8-32 weeks in male senescence-accelerated mouse prone 10 (SAMP-10). They were subjected to the T-maze test, followed by immunohistochemical analysis, to assess the influence of the shaking exercise on the M1 muscarinic acetylcholine receptor (CHRM1) and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) of the dorsal hippocampus and medial prefrontal cortex (dHC-mPFC). RESULTS: The T-maze test demonstrated that the shaking group had less hesitation in the face of selecting direction at week 24. In the immunohistochemical analysis, more CHRM1s were in the CA3 subregion and more AMPARs were in the subiculum. CHRM1s and AMPARs were maintained in the CA1 region and the mPFC. The CHRM1s seem to have a positive effect on the AMPAR in the dentate gyrus (DG) region and the CA3 region. In the CA1 region, CHRM1s were negatively correlated with AMPARs. In addition, high-density neurons were expressed in the shaking group in the upstream DG, the middle part and the distal part of CA3, the distal part of CA1, and the mPFC. CONCLUSIONS: Our results raise the possibility that maintenance of the spatial working memory effect observed with the shaking exercise is driven in part by the uneven affection of CHRM1s and AMPARs in the dHC-mPFC circuit system and significantly maintains the neuronal expression in the dHC-mPFC.

INTRODUCTION: Although exercise can prevent cognitive decline due to aging, few elderly individuals are able to exercise for long. Therefore, an exercise method for older adults that is feasible for a long duration without overexertion is necessary. In this study, we focused on exercise by shaking. This study examined the possibility to prevent the decline in memory through regular and long-term shaking exercise using a senescence-accelerated mouse (SAM) model. Behavioral analysis was conducted, and histological changes in the mouse brain were examined to evaluate whether this stimulation method could become a novel exercise method. MATERIALS AND METHODS: The shaking exercise was applied to SAMP10 mice for 30 min 3 times per week for 25 continuous weeks. Behavioral analysis included a step-through passive avoidance test, whereas the histological analysis involved immunohistochemical staining using the anti-glutamate receptor (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors [AMPAR]) antibody in the hippocampus. The number and area of nerve cells in the hippocampal regions were measured and compared between groups. RESULTS: Behavioral analysis revealed that the shaking group retained memory longer than the control group, and memory capacity decline was suppressed. Additionally, histological examination showed that the shaking group had a higher number of AMPAR receptor-positive neurons per area in the hippocampal CA1 and CA3 regions than the control group, suggesting that degeneration and shedding of neurons due to aging was suppressed. DISCUSSION/CONCLUSION: We believe that shaking could become an exercise therapy that can reduce the decline in memory with aging and expect its human application in the future.

Patients with lower limb amputation experience "embodiment" while using a prosthesis, perceiving it as part of their body. Humans control their biological body parts and receive appropriate information by directing attention toward them, which is called body-specific attention. This study investigated whether patients with lower limb amputation similarly direct attention to prosthetic limbs. The participants were 11 patients with lower limb amputation who started training to walk with a prosthesis. Attention to the prosthetic foot was measured longitudinally by a visual detection task. In the initial stage of walking rehabilitation, the index of attention to the prosthetic foot was lower than that to the healthy foot. In the final stage, however, there was no significant difference between the two indexes of attention. Correlation analysis revealed that the longer the duration of prosthetic foot use, the greater the attention directed toward it. These findings indicate that using a prosthesis focuses attention akin to that of an individual's biological limb. Moreover, they expressed that the prosthesis felt like a part of their body when they could walk independently. These findings suggest that the use of prostheses causes integration of visual information and movement about the prosthesis, resulting in its subjective embodiment.

Introduction: Patients with dementia show reduced adaptive, behavioral, and physiological responses to environmental threats. Physical exercise is expected to delay brain aging, maintain cognitive function and, consequently, help dementia patients face threats and protect themselves skillfully. Methods: To confirm this, we aimed to investigate the effects of the shaking exercise on the avoidance function in the senescence-accelerated mouse-prone strain-10 (SAMP-10) model at the behavioral and tissue levels. SAMP-10 mice were randomized into 2 groups: a control group and a shaking group. The avoidance response (latency) of the mice was evaluated using a passive avoidance task. The degree of amygdala and hippocampal aging was evaluated based on the brain morphology. Subsequently, the association between avoidance response and the degree of amygdala-hippocampal aging was evaluated. Results: Regarding the passive avoidance task, the shaking group showed a longer latency period than the control group (p < 0.05), even and low intensity staining of ubiquitinated protein, and had a higher number of and larger neurons than those of the control group. The difference between the groups was more significant in the BA region of the amygdala and the CA1 region of the hippocampus (staining degree: p < 0.05, neuron size: p < 0.01, neuron counts: p < 0.01) than in other regions. Conclusions: The shaking exercise prevents nonfunctional protein (NFP) accumulation, neuron atrophy, and neuron loss; delays the aging of the amygdala and hippocampus; and maintains the function of the amygdala-hippocampal circuit. It thus enhances emotional processing and cognition functions, the memory of threats, the skillful confrontation of threats, and proper self-protection from danger. (C) 2021 The Author(s) Published by S. Karger AG, Basel

To execute the intended movement, the brain directs attention, called body-specific attention, to the body to obtain information useful for movement. Body-specific attention to the hands has been examined but not to the feet. We aimed to confirm the existence of body-specific attention to the hands and feet, and examine its relation to motor and sensory functions from a behavioral perspective. The study included two groups of 27 right-handed and right-footed healthy adults, respectively. Visual detection tasks were used to measure body-specific attention. We measured reaction times to visual stimuli on or off the self-body and calculated the index of body-specific attention score to subtract the reaction time on self-body from that off one. Participants were classified into low and high attention groups based on each left and right body-specific attention index. For motor functions, Experiment 1 comprised handgrip strength and ball-rotation tasks for the hands, and Experiment 2 comprised toe grip strength involved in postural control for the feet. For sensory functions, the tactile thresholds of the hands and feet were measured. The results showed that, in both hands, the reaction time to visual stimuli on the hand was significantly lesser than that offhand. In the foot, this facilitation effect was observed in the right foot but not the left, which showed the correlation between body-specific attention and the normalized toe gripping force, suggesting that body-specific attention affected postural control. In the hand, the number of rotations of the ball was higher in the high than in the low attention group, regardless of the elaboration exercise difficulty or the left or right hand. However, this relation was not observed in the handgripping task. Thus, body-specific attention to the hand is an important component of elaborate movements. The tactile threshold was higher in the high than in the low attention group, regardless of the side in hand and foot. The results suggested that more body-specific attention is directed to the limbs with lower tactile abilities, supporting the sensory information reaching the brain. Therefore, we suggested that body-specific attention regulates the sensory information to help motor control.

Introduction:The disabling effects of dementia, an incurable disease with little effect on mortality, affect society far more than many other conditions.Objective:The aim of this study was to stop or delay the onset of dementia using low-cost methods such as physical exercise.Methods:Senescence-accelerated model-prone (SAMP) 10 mice were made to perform a user-friendly shaking exercise for 25 weeks. The motor function and hippocampal functions (learning, spatial cognition) of the mice were evaluated using behavioral experiments. The degree of hippocampal aging was evaluated based on brain morphology. The association between behavioral performance of the mice and the degree of hippocampal aging was then evaluated.Results:The behavioral test results showed that the shaking group had higher motor coordination (p< 0.01) and motor learning (p< 0.05). Significantly higher performances in the learning ability were observed in the shaking group at a middle-period experiment (p< 0.05); the spatial cognitive functions also improved (p< 0.05). The shaking group showed delayed ageing of cells in the dentate gyrus (DG; area:p< 0.01) and cornu Ammonis (CA; area:p< 0.01) regions of the hippocampus.Conclusions:The shaking exercise enhances the activity of mice and reduces age-associated decreases in learning and spatial cognitive functions. Regarding hippocampal morphology, shaking exercise can prevent non-functional protein accumulation, cell atrophy, and cell loss. Specifically, shaking exercise protects cell growth and regeneration in the DG area and enhances the learning function of the hippocampus. Furthermore, shaking exercise maintained the spatial cognitive function of cells in the CA3 and CA1 regions, and prevented the chronic loss of CA2 transmission that decreased the spatial memory decline in mice.

本学では、臨床検査技師教育課程において人体解剖実習を導入し、その有効かつ有益な教育方法を考察した。その結果、「心臓の構造を理解できた」と回答した学生は96%であり、他臓器においても80%以上と高率であった。また、医療職として必須の倫理的教育効果についても「ご遺体の尊厳についての理解」に関して96%の学生から肯定的な回答が得られた。一方で、教員の不足等による不満もあったが、今後は大学院生や勉学の意識の強い卒業生の実習参加などを通して、より広くかつ意義深い実習にしていきたいと考えている。(著者抄録)

著者らは、骨粗鬆症に伴う骨折の予防を目的に、振動刺激と振盪刺激の2種類の刺激を組み合わせたコンビネーション刺激装置を開発した。今回、骨密度低下モデルマウスを用いて、この新規刺激装置の効果を検討した。8週齢の雌性マウス12匹に対して卵巣摘出により骨密度低下モデルマウスを作製した後、刺激群と非刺激群に分け、刺激群には連続10週間に亘り刺激を行った。類骨量、類骨面、類骨幅、類骨厚、骨石灰化速度、組織を基準とした骨形成速度は、刺激群が非刺激群に比べて有意に高かった。骨梁間隙は、刺激群が非刺激群に比べて低値を示した。また、大腿四頭筋における骨形成タンパク質2、IL-1β、MyoDなどの発現解析においても、刺激群が非刺激群に比べて有意に高い値を示した。本刺激装置は、骨密度低下モデルマウスに対して骨形成を促進させ、骨密度の低下を予防できる可能性が示唆された。

A病院で日常的に患者の生活援助にかかわっている看護師922名を対象に、解剖生理学の知識活用の困難に、看護師の背景による違いがあるかについて質問紙調査を実施した。対象看護師は、臨床経験年数3年以内、4から5年目、6から10年目、11から15年目、16年以上の5群に分けて背景の検討を行った。400名から回答が得られ、391名(男性48名、女性343名)の有効回答を分析対象とした。解剖生理学の知識活用の困難の違いは「臨床経験年数」の差で見られた。「解剖生理学は苦手である」「解剖生理学で活用されている用語そのものが難しい」などの5項目で特に臨床経験年数での違いが見られた。「解剖生理学は苦手である」「解剖生理学で使われている化学式が難しい」「解剖生理学で使われている分子レベルの知識が難しい」の3項目では、臨床経験年数の多い看護師が自分より経験の少ない看護師よりも困難を感じていることが多いという結果が得られた。

In Japan, 13 million people have osteoporosis, including approximately 9 hundred thousand people who are bedridden owing to bone fractures from falls. Preventing osteoporosis is considered to be an important and effective way of preventing fall-related fractures. Thus, we developed a novel method of locomotor stimulation and analyzed its effectiveness in mice. Specifically, we created a double-loading device that combines vibration and shaking stimulation. The device was used to continuously stimulate ovariectomy-induced decreased bone density mouse models 30 minutes daily for 10 weeks. We then collected femur samples, created undecalcified tissue slices, calculated parameters using bone histomorphomtry, and conducted comparative testing. BS/TV (bone surface/tissue volume), N.Oc/ES (osteoclast number/eroded surface), Oc.S/ES (osteoclast osteoid surface/eroded surface), Omt (osteoid maturation time), Tb.N (trabecular number), Mlt (mineralization lag time) < (p < 0.01), N.Ob (osteoblast number), N.Ob/TV (osteoblast number/tissue volume), sLS (single labeled suface), N.Mu.Oc/ES (multinucle osteoclast number/eroded surface), and N.Mo.Oc/ES (mononucle osteoclast number/eroded surface) (p < 0.05) were significantly higher in the stimulation group than in the non-stimulation group. In addition, BS/BV (bone surface/bone volume), Tb.Sp (trabecular separation), MAR (mineral apposition rate), Aj.Ar (adjusted apposition rate) (p < 0.01), ES (eroded surface ), ES/BS (eroded surface/bone surface), and BRs.R (bone resorption rate) (p < 0.05) were significantly lower in the stimulation group than in the non-stimulation group. These results suggest that stimulation activated osteoblasts and osteoclasts, thereby leading to highly active bone remodeling. We anticipate that bone mineralization will subsequently occur, suggesting that this stimulation technique is effective in preventing osteoporosis by alleviating sudden bone density loss.

[Purpose] The number of bedridden patients requiring nursing care in Japan has increased sharply in recent years because of its aging population and advances in medical care and has become a major social issue. Because bedridden patients are susceptible to nursing and healthcare-associated pneumonia, it is very important to improve their immunocompetence. Therefore, the effect of exercise therapy on stimulation of cytokine secretion in the saliva of bedridden patients was investigated. [Subjects and Methods] The subjects of this study were bedridden patients admitted to nursing care facilities. They were instructed to perform active assistive movement in the supine and sitting positions, with vital signs used as an index of the exercise load. Thirty-five patients fulfilled the inclusion criteria, which included cerebrovascular disease as the main cause of being bedridden and at least 6 months since onset. Interleukins were measured by enzyme-linked immunosorbent assay as immune mediators. [Results] Vital signs improved significantly after therapeutic exercise intervention, and the IL-6, IL-8, IL-15, and IL-17 levels also increased significantly after the intervention. [Conclusion] The results demonstrated that measurement of saliva samples may offer a safe minimally invasive method of measuring immune response in bedridden patients. This study suggests that exercise therapy may hold promise as an effective means of improving immunity in bedridden patients and may contribute to preventing aspiration pneumonia and promoting spontaneous recovery.

Recently, health awareness in Japan has been increasing and active exercise is now recommended to prevent lifestyle-related diseases. Cytokine activities have many positive effects in maintaining the health of a number of organs in the body. Myokines are cytokines secreted by skeletal muscles in response to exercise stimulation, and have recently generated much attention. Around 700,000 patients in Japan suffer from rheumatoid arthritis, making it the most prevalent autoimmune disease that requires active prevention and treatment. In the present study, a mouse model of spontaneous arthritis (SKG/Jcl) was subjected to continuous exercise stimulation, starting before the disease onset, to examine the effects of anti-inflammatory and inflammatory cytokine secretion on arthritis. For this stimulation, we developed a device that combines shaking and vibration. The results revealed that exercise stimulation delayed the onset of arthritis and slowed its progression. Thickened articular cartilage and multiple aggregates of chondrocytes were also observed. Further, exercise stimulation increased the expression of IL-6, IL-10, and IL-15, and inhibited TNF-α expression. From these results, we infer that the anti-inflammatory effects of IL-6 and IL-10, which showed increased expression upon exercise stimulation, inhibited the inflammatory activity of TNF-α and possibly delayed the onset of arthritis and slowed its progression. Novel methods for preventing and treating arthritis under clinical settings can be developed on the basis of these findings.

In order to elucidate the function of anti-aromatic acid decarboxylase (AADC)-only-positive cells in the alimentary canal, 5-hydroxy-L-tryptophan (5-HTP) or L-3,4-dihydroxyphenylalanine (L-DOPA) was intraperitoneally injected into the laboratory shrew, Suncus murinus, and immunohistochemical studies were conducted on continuous sections of the alimentary canal using specific antisera against tyrosine hydroxylase (TH), AADC, dopamine (DA), and serotonin (5-HT). AADC-only-positive cells localized to the epithelial layer of the alimentary canal from the stomach to the large intestine. These AADC-only-positive cells became DA- and AADC-positive cells after L-DOPA injection, and 5-HT- and AADC-positive cells after 5-HTP injection. These results strongly indicate that the AADC-only-positive cells in the alimentary canal of Suncus murinus are capable of synthesizing DA and 5-HT simultaneously upon administration of L-DOPA and 5-HTP.

Osteoporosis is leaving bones more fragile and susceptible to fracture. It has a massive impact, both physically and mentally, markedly diminishing quality of life. A new form of therapeutic exercise or physical therapy that mitigates the abrupt decrease in bone density in postmenopausal women must quickly be developed to avoid those problems. In this study, ovariectomy (OVX) mice were used as models to simulate the decrease in bone density observed in postmenopausal women. Physical therapy via a shaking stimulus, in the form of moving a platform that rotates in a roughly circular motion in the horizontal plane, was studied as a way to prevent the decrease in bone density of the lumbar vertebrae by analysis of bone histomorphometry, a feat that the stimulus from conventional therapeutic exercise and physical therapy have failed to achieve. Comparison of the stimulus/ovariectomized (+/+) group with the -/+ group indicated significant increases in ES (P < 0.01), N. Mu. Oc (P < 0.05), OV (P < 0.05), O. Th (P < 0.01), and L. Th (P < 0.01) in the +/+ group. If this finding is used clinically, we believe that it could lead to therapy that would prevent compression fractures of the lumbar vertebrae.

Background and aims Elderly individuals who suffer a fracture develop a gait disturbance and require prolonged bedrest. A fracture has a massive impact both physically and mentally and markedly diminishes quality of life. A new form of therapeutic exercise that mitigates the abrupt decrease in bone density in postmenopausal women must soon be developed so that those problems can be avoided.Methods The current study used a model of the decrease in bone density in ovariectomized mice to simulate postmenopausal women. The stimulus was provided by a shaking horizontal platform rotating in a circular motion.Results Comparison of the +/+ (ovariectomized/stimulated) group and +/- group indicated a significant decrease in BV/TV (p < 0.01), Tb.Th (p < 0.01), and Tb.N (p < 0.05) in the +/+ group and a significant increase in OV/BV (p < 0.01), OV/ OS (p < 0.01), BFR/BV (p < 0.01), dLS/BS (p < 0.05), MS/BS (p < 0.05), BRs.R (p < 0.01), and Tb.Sp (p < 0.01) in the +/+ group. Physical therapy to prevent a decrease in bone density was studied via stimulus in the form of rotation of a platform. Analysis of bone histomorphometry revealed lessening of the decrease in bone density of the lumbar vertebrae, a feat that the stimulus from conventional physical therapy had failed to achieve.Conclusion The current study delivered a shaking stimulus to mice in a model of postmenopause. Analysis of bone histomorphometry of the lumbar vertebrae suggested lessening of the abrupt decrease in bone density of trabecular bone. If this finding is used clinically, it could lead to physical therapy exercise that would be able to prevent compression fractures of the lumbar vertebrae.

After finding tonsil-like structures near the entrance of vagina of a laboratory shrew (Suncus murinus), which we subsequently designated as vaginal tonsils, we performed detailed immunohistochemical and developmental studies. The location of T and B cells in the vaginal tonsils differed from that in the palatine tonsils or that in the lymphoid nodes of other animals. The boundary between the germinal center region and the region encompassing follicular interfollicular tissue was not clear. B cells were widely distributed and very dense in the parenchyma, but they were scattered in the epithelial area (B cells were present in around 90% of the vaginal tonsil tissue). In contrast, T cells were scattered in the parenchyma and in the epithelial area (T cells were present in around 10% of the vaginal tonsil tissue). B cells were more prominent than T cells throughout the development of these structures and the epithelium was invaded by many immigrating cells. The size of the vaginal tonsils changed during postnatal development. Vaginal tonsils are structurally similar to other tonsils, and they may function to protect the vagina from infection.

The events that take place during the prophase of meiosis I are essential for the correct segregation of homologous chromosomes. Defects in these processes likely contribute to infertility or recurrent pregnancy loss in humans. To screen for candidate genes for reproductive failure due to meiotic defects, we have analyzed the gene expression patterns in fetal, neonatal and adult gonads of both male and female mice by microarray and thereby identified 241 genes that are expressed specifically during prophase of meiosis I. Combined with our previous data obtained from developing spermatocytes, a total of 99 genes were identified that are upregulated in early prophase I. We confirmed the meiotic prophase I-specific expression of these genes using qRT-PCR. To further screen this panel for candidate genes that fulfill important roles in homologous pairing, synapsis and recombination, we established a gene transfer system for prophase I oocytes in combination with in vitro organ culture of ovaries, and successfully determined the localization of the selected genes. This gene set can thus serve as a resource for targeted sequence analysis via next-generation sequencing to identify the genes associated with human reproduction failure due to meiotic defects. Journal of Human Genetics (2012) 57, 515-522; doi: 10.1038/jhg.2012.61; published online 31 May 2012

Background: Cumulative evidence suggests that DNA secondary structures impact DNA replication, transcription and genomic rearrangements. One of the best studied examples is the recurrent constitutional t(11;22) in humans that is mediated by potentially cruciform-forming sequences at the breakpoints, palindromic AT-rich repeats (PATRRs). We previously demonstrated that polymorphisms of PATRR sequences affect the frequency of de novo t (11;22)s in sperm samples from normal healthy males. These studies were designed to determine whether PATRR polymorphisms affect DNA secondary structure, thus leading to variation in translocation frequency.Methods: We studied the potential for DNA cruciform formation for several PATRR11 polymorphic alleles using mobility shift analysis in gel electrophoresis as well as by direct visualization of the DNA by atomic force microscopy. The structural data for various alleles were compared with the frequency of de novo t(11; 22) s the allele produced.Results: The data indicate that the propensity for DNA cruciform structure of each polymorphic allele correlates with the frequency of de novo t(11; 22) s produced (r = 0.77, P = 0.01).Conclusions: Although indirect, our results strongly suggest that the PATRR adopts unstable cruciform structures during spermatogenesis that act as translocation hotspots in humans.

Han: SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated &quot;Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation,&quot; &quot;LPS/IL-1-Mediated Inhibition of RXR Function,&quot; and &quot;Liver X Receptor (LXR)/RXR Activation.&quot; These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat. Copyright © 2011 the American Physiological Society.

Kugita M, Nishii K, Morita M, Yoshihara D, Kowa-Sugiyama H, Yamada K, Yamaguchi T, Wallace DP, Calvet JP, Kurahashi H, Nagao S. Global gene expression profiling in early-stage polycystic kidney disease in the Han: SPRD Cy rat identifies a role for RXR signaling. Am J Physiol Renal Physiol 300: F177-F188, 2011. First published October 6, 2010; doi:10.1152/ajprenal.00470.2010.-Han: SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," " LPS/IL-1-Mediated Inhibition of RXR Function," and " Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.

The constitutional t(11;22) is the most frequent recurrent non-Robertsonian translocation in humans, the breakpoints of which are located within palindromic AT-rich repeats on 11q23 and 22q11 (PATRR11 and PATRR22). Genetic variation of the PATRR11 was found to affect de novo t(11;22) translocation frequency in sperm derived from normal healthy males, suggesting the hypothesis that polymorphisms of the PATRR22 might also influence the translocation frequency. Although the complicated structure of the PATRR22 locus prevented determining the genotype of the PATRR22 in each individual, genotyping of flanking markers as well as identification of rare variants allowed us to demonstrate an association between the PATRR22 allele type and the translocation frequency. We found that size and symmetry of the PATRR22 affect the de novo translocation frequency, which is lower for the shorter or more asymmetric versions. These data lend support to our hypothesis that the PATRRs form secondary structures in the nucleus that induce genomic instability leading to the recurrent translocation.

Prophase I of male meiosis during early spermatogenesis involves dynamic chromosome segregation processes, including synapsis, meiotic recombination and cohesion. Genetic defects in the genes that participate in these processes consistently cause reproduction failure in mice. To identify candidate genes responsible for infertility in humans, we performed gene expression profiling of mouse spermatogenic cells undergoing meiotic prophase I. Cell fractions enriched in spermatogonia, leptotene/zygotene spermatocytes or pachytene spermatocytes from developing mouse testis were separately isolated by density gradient sedimentation and subjected to microarray analysis. A total of 726 genes were identified that were upregulated in leptotene/zygotene spermatocytes. To evaluate the screening efficiency for meiosis-specific genes, we randomly selected 12 genes from this gene set and characterized each gene product using reverse transcription (RT)-PCR of RNA from gonadal tissues, in situ hybridization on testicular tissue sections and subcellular localization analysis of the encoded protein. Four of the 12 genes were confirmed as genes expressed in meiotic stage and 2 of these 4 genes were novel, previously uncharacterized genes. Among the three confirmation methods that were used, RT-PCR appeared to be the most efficient method for further screening. These 726 candidates for human infertility genes might serve as a useful resource for next-generation sequencing combined with exon capture by microarray. Journal of Human Genetics (2010) 55, 293-299; doi:10.1038/jhg.2010.26; published online 26 March 2010

ヒトは長時間立った状態を維持する際に、無意識に体の重心を左右に移動させて疲労を抑制する。これは穏やかな全身運動を行っている状態であるといえる。振動板の上で静止立位を維持することは、静止した場所に立つよりより効果的な全身運動を誘導すると考えられる。振動刺激が骨密度減少に与える効果について調べた。骨ミネラル密度(BMD)減少モデルマウスに振動刺激を与えると、大腿骨強度の減少が抑制された。骨の部位により抑制の程度に差がみられた。BMD減少抑制効果は骨の形態、組成、物理的分析により確認された。この方法はヒトにおいても有用であることが示唆された。振動刺激による全身運動は高齢者のみでなく若年者に対しても、骨折予防および健康促進における理学療法として有用であると考えられた。

振盪刺激は閉経後に起こる骨密度低下を防止する効果的な物理療法と考えられている。大腿骨を補強する筋群は、振盪刺激により骨に間接的影響を与えている。この刺激は下半身の筋を中心に等尺性運動もしくは等張性運動を強制的に行う。本研究は下半身の運動に重要とされ、またインナーマッスルとしてよく知られている大腰筋について解析を行った。筋の発生や再生および分化に影響を与える特異的なタンパク質の発現量をWestern blot法を用いて組織学的解析と合わせて比較検討を行った。実験は、ICRマウスを使用し、卵巣摘出した群と正常群の2群に分類し、それぞれの群に対して、刺激を行った群と行わない群に区分した(卵巣摘出/振盪：+/+, +/-,-/+,-/-)、計4群に対して実験を行った。振盪刺激を10週間継続した後に大腰筋を採取し、起始側と停止側の２つの領域を解析するため２等分した。組織学的解析は、筋線維横断面をH.E.染色し、筋線維の短径と線維数を計測した。組織学的解析において、振盪刺激による筋線維の肥大は、筋線維短径の計測により停止側で確認された。しかし、筋線維数の変化については顕著な違いは認められなかった。筋の肥大を促進するタンパク質であるgrowth differentiation factor 8 (GDF-8)と発生・分化に関与するmyogenic factor 5 (Myf-5)、myogenic factor 6 (Myf-6)のタンパク質発現量は、刺激を行った群と行わなかった群において行った群の停止側に増加傾向を示した。大腰筋のような二関節筋の働きをする筋において起始・停止部分で筋肥大ならびに発生や分化に部位差の相違が認められた。本研究において大腰筋の停止部で刺激による部位差が顕著であったことから振盪刺激が姿勢安定による転倒予防と下半身、特に大腿部における骨を補強する効果的な物理療法になり得ると考える。

Venipuncture is a routine procedure performed at medical institutions for blood collection and blood donation, as well as for health screening and testing. Venipuncture is invasive and usually mildly painful, but it occasionally causes internal bleeding and chronic persistent pain, which is referred to as complex regional pain syndrome. The most common site of venipuncture is the upper arm. The present study macroscopically and anatomically ascertained positional relationships between cutaneous nerves and veins in the cubital (aka antecubital) fossa in many cadaveric dissections to determine the risk of peripheral nerve injury during venipuncture. We identified the most suitable venipuncture site in the upper arm. The medial cutaneous nerve of the forearm (MCNF) passed above the median cubital vein (MCBV) in 27 of 128 cases (21.1%), and was located inferior to the MCBV in 37 of 128 cases (28.9%). The MCBV also passed above the lateral cutaneous nerve forearm (LCNF) in 8 of 128 cases (6.2%). The LCNF was located deeper than the MCBV in 56 of 128 cases (43.8%). The distribution of cutaneous veins and nerves widely varies, and while no single area suitable for all individuals was identified, puncture of the MCBV near the cephalic vein is the least likely to cause nerve damage.

Venipuncture is a routine procedure performed at medical institutions for blood collection and blood donation, as well as for health screening and testing. Venipuncture is invasive and usually mildly painful, but it occasionally causes internal bleeding and chronic persistent pain, which is referred to as complex regional pain syndrome. The most common site of venipuncture is the upper arm. The present study macroscopically and anatomically ascertained positional relationships between cutaneous nerves and veins in the cubital (aka antecubital) fossa in many cadaveric dissections to determine the risk of peripheral nerve injury during venipuncture. We identified the most suitable venipuncture site in the upper arm. The medial cutaneous nerve of the forearm (MCNF) passed above the median cubital vein (MCBV) in 27 of 128 cases (21.1%), and was located inferior to the MCBV in 37 of 128 cases (28.9%). The MCBV also passed above the lateral cutaneous nerve forearm (LCNF) in 8 of 128 cases (6.2%). The LCNF was located deeper than the MCBV in 56 of 128 cases (43.8%). The distribution of cutaneous veins and nerves widely varies, and while no single area suitable for all individuals was identified, puncture of the MCBV near the cephalic vein is the least likely to cause nerve damage.

Regions containing palindromic sequence are known to be susceptible to genomic rearrangement in prokaryotes and eukaryotes. Palindromic AT-rich repeats (PATRR) are hypervariable in the human genome, manifesting size polymorphisms and a propensity to rearrange. Size variations are mainly the result of internal deletions, while two PATRRs on 11q23 and 22q11 (PATRR11 and 22) contribute to generation of the t(11;22), a recurrent constitutional translocation. In this study, we analyzed the PATRR11 sequence of numerous polymorphic alleles in detail. Various types of shorter variants are likely derived from the most frequent similar to 450 bp PATRR11 by deletion. Deletion variants possess a significant number of identical nucleotides at their two endpoints, indicating the possible involvement of direct repeats within the PATRR11. Rare variants with insertional alterations involve AT-rich sequences of unknown origin. This is in contrast to palindrome-mediated translocations between PATRRs that manifest smaller deletions and only a limited number of identical nucleotides at the breakpoints. Further, we identified a rare translocation product that has a non-AT-rich insertion of a transcribed gene segment at the translocation breakpoint. Our data suggest that the outcomes of palindrome-mediated re-arrangements reflect distinct molecular pathways; intra-palindrome re-arrangements are possibly dictated by a replication slippage or microhomology-directed repair pathway, and inter-palindrome translocations are likely driven by non-homologous end joining.

We analyzed de novo constitutional t(11;22) translocation frequency in sperm derived from normal healthy males as a function of the age of the sperm donors (from 25 to 51). Translocation-specific polymerase chain reaction demonstrated no age-dependent increment in the frequency of the rearrangements.

Several reports on different in vitro and in vivo models have revealed neuroprotective effects afforded by nicotine treatment. Nicotine is a cognitive enhancer that acts by increasing vigilance and improving learning and memory. We previously reported that nicotine withdrawal up-regulated transcription of some immediately early genes (IEGs), apoptosis-related genes, and signal transduction-related genes in cultures of pheochromocytoma PC12 cells, which are of neuronal lineage (Ichino et al., 1999; Ichino et al., 2002; Yamada et al., 2005). Especially, nicotine withdrawal increased the levels of Bag-1, Rab2, Hsp70, and Raf1 proteins in the cerebral cortex, hippocampus, and striatum in vivo in mice. Egr-1 and Nur77 protein levels were dramatically increased by nicotine withdrawal. (Yamada et al., 2006). Also, there is a study that analyzed the expression of mRNAs by in situ hybridization using a rat genome array after acute intermittent nicotine treatment and found that some mRNA levels of them (Nr4a1, Egr-1 and Egr-2) are significantly up-regulated in the rat cerebral cortex and hippocampal regions (Belluardo et al., 2005). In the present study, we aimed at further elucidating the effects of nicotine withdrawal on the expression of the upstream proteins of IEGs in the brain of mice after chronic administration of nicotine. We observed significant increases in the protein levels of Egr-1 and Nur77 in the specific regions and in specific cells in the mouse brain by Western blot analysis and by immunohistochemistry. The present results suggest that such changes may also occur in the brain of smokers during abstaining from smoking and may be related to some of their symptoms of nicotine withdrawal. © Society of Integrated Sciences.

77 CAGリピートを有するヒトhuntingtinノックインマウスを用いて、ニューロン特異的huntingtin集積の特徴を免疫組織化学的に調べた。その結果、異型接合体マウスでは、huntingtin N末端断片の集積は線条体突起ニューロンの核と神経網において特異的に形成され、この集積形成は加齢に従って進行し、蛋白分解又はシャペロン蛋白と相互作用し、側坐核において最も顕著に生じた。これらの変異マウスは異常な攻撃行動を示した。同型接合体マウスでは、核内及び神経網に重い沈着物が検出され、それは他の部位に広がり、特徴的な大きな核周囲部集積を認めた。これらの変異マウスにおいて線条体や関連ニューロンの細胞死は認めなかった。以上より、ポリグルタミン集積は神経細胞死と必ずしも関連せず、これらのヒトhuntingtinノックインマウスはハンチントン病の治療アプローチに有用と考えられた。

Background. It has been well documented that two factors, brain death (131)) and ischemia/reperfusion (I/R) injury, have distinct but overlapping adverse influences on the clinical outcome of renal transplantation.Method. We previously established a rat model of renal isografting from brain dead donors. In the present study, we performed genomic expression profiling with a high-density oligonucleotide microarray to identify genes that were upregulated or downregulated by BD and/or I/R injury.Results. Among a total of 20,550 genes, most of those upregulated by BD were genes for adhesion molecules and cytokines or for chemokines such as Gro1 and IP-10. When overexpression of these genes was assessed by real-time reverse transcriptase-polymerase chain reaction, it was only observed one hr after the engraftment of kidneys from BD donors and returned to baseline thereafter, indicating the presence of an acute systemic inflammatory response to BD. Analysis of biologic networks demonstrated the activation of specific pathways that were clearly different for BD and I/R injury. The p53 and NF kappa B pathway was involved in the acute response to BD, whereas the Myc, Jun, and c-fos pathway was involved in I/R injury. Investigation of secretory protein genes identified LCN2 and SPP1 as candidate genes for biologic markers.Conclusion. Because our experimental system is a good model of renal transplantation from brain dead or living human donors, our data may be useful for elucidating the pathologic processes involved and for identification of novel markers for graft dysfunction of renal transplantation.

We isolated BAC clones that cover the entire hox gene loci in the medaka fish Oryzias latipes. The BAC clones were characterized by the Southern hybridization with many hox gene probes isolated in our previous study and by PCR using primers designed for selective amplification of respective hox genes. Then, the BAC clones have been subjected to shotgun sequencing. The results revealed the organization of the entire hox gene loci. Forty-six hox genes in total are encoded in seven clusters as follows: 10 hox genes in Aa cluster; 5 in Ab; 9 in Ba; 4 in Bb; 10 in Ca; 6 in Da; and 2 in Db. Together with the information on the hox gene loci registered in the Fugu genome database and in the Danio genome database, the physical maps of three fish genomes were constructed and compared one another. Not only numbers of hox genes but also the distances between the neighboring hox genes are highly similar between medaka and fugu. As for six clusters, Aa, Ab, Ba. Bb, Ca and Da that are commonly present in the three fishes, only few or no differences were found in each cluster. Thus, the hox gene sets should have been well conserved once they had been established in respective species. (c) 2005 Elsevier B.V All rights reserved.

Palindromic sequences are dispersed in the human genome and may cause chromosomal translocations in humans. They constitute unsequenced gaps in the human genome because of their resistance to PCR amplification, cloning into vectors, and sequencing. We have overcome these difficulties by using a combination of optimized PCR conditions, cloning in a recombination,deficient E. coli strain, and RNA polymerases in sequencing. Using these methods, we analyzed a palindromic AT-rich repeat (PATRR) in the neurofibromatosis type I (NF1) gene on chromosome 17 (17PATRR). The 17PATRR manifests a size polymorphism due to a highly variable length of (AT) (n) dinucleotide repeats within the PATRR. 17PATRRs can be categorized into two types: a longer one that comprises a nearly or completely perfect palindrome, and a shorter one that represents its deleted asymmetric derivative. In vitro analysis shows that the longer 17PATRR is more likely to form a cruciform structure than the shorter one. Two reported t(17;22)(q11;q11) patients with NF1, whose breakpoints were identified within the 17PATRR, have translocations that are derived from perfect or nearly perfect palindromic alleles. This implies that the symmetric structure of a PATRR can induce a translocation. We identified conserved PATRRs within the NF1 gene in great apes and similar inverted repeats in two Old World monkeys, but not in New World monkeys or other mammals. This indicates that the palindromic region appeared approximately 25 million years ago and elongated during primate evolution. Although such palindromic regions are usually unstable and disappear rapidly due to deletion, the 17PATRR in the NF1 gene was stably conserved during evolution for reasons that are still unknown.

We previously reported that nicotine withdrawal up-regulates transcription of some immediately early genes (IEGs), c-fos (Ichino et al., 1999) and egr1, nur77 (Ichino et al., 2002) in cultures of pheochromocytoma PC12 cells, which are of neuronal lineage. In the present study we aimed at further elucidating the effects of nicotine withdrawal on the expression of the genes downstream of IEGs. We examined the changes in the protein levels of 2 GTP-binding proteins, Rab2 (Ras-related protein) and Rac1. PC12 cells were cultured in the presence of nicotine for 24 hours, and then the nicotine was removed from the medium. The protein level of Rab2 was low in the presence of nicotine, but was rapidly increased after nicotine withdrawal. In contrast, that of Rac1 did not change after the withdrawal. Considering the neuroprotective effect of nicotine, we also examined the level of Bag-1 protein, which is a binding protein for Bcl-2, an anti-apoptotic factor, and found a slight increase in the gene expression of Bag-1 following nicotine withdrawal. Among 56-kDa, 50-kDa, and 36-kDa protein components of the Bag-1 protein complex, the levels of 56-kDa and 50-kDa proteins were not changed by the addition or withdrawal of nicotine; but the level of the 36-kDa protein, which had been increased in the presence of nicotine, was markedly decreased after nicotine withdrawal. The present results suggest that such changes may also occur in individuals during abstaining from smoking and be related to the withdrawal symptoms experienced after smokers stop smoking.

医療系学生のための解剖学実習に供された20遺体中4例において,腎臓に出入りする血管系の顕著な変異例が発見された.遺体へ10%ホルマリン液を大腿動脈から注入し,脳を錐体交叉の部位で切断後摘出した.腎副動脈は3例に出現し,腎門外で動脈枝が分節状に一部配列し,腎実質内へ進入したものと考えられた.腎副静脈は右側のみに2例出現し,左側には見られなかった.左腎静脈が腹大動脈の背側を通り,左第3腰静脈ならびに第4腰静脈に合流のする例が1例みられた.また,腎盤が上・下の2個に分割し,尿管が上・下の2本存在する例が動・静脈の変異と同時に1例観察した.腎盤が2個に分割した際には血管分布も1分節増加するように見られた

Background: Brain death (BD) and the subsequent ischemia/reperfusion (I/R) injury have cardinal implications for the pathogenesis of kidney transplantation (Tx). However, the precise mechanistic pathway of BD and the subsequent I/R injury are unknown. In this study, we performed genome-wide analysis for differential gene expression in kidney isografts from BD donors. Their gene expressions were compared with those from living sources. Methods: Kidneys from BD rats were engrafted and their gene expressions were compared with those from living controls. Donors were intubated, and mechanically ventilated for 6 hours. Grafts were harvested 6 hours after BD, and 1 hour after engraftment. The expression profile of approximately 20,500 genes was analyzed. Results: Gene expression of chemokines (Scya2 and Gro1), cytokines (IL-1 and -6) and adhesion molecules (E- and P-selectin and ICAM-1) were upregulated in the BD kidneys and 1 hour after engraftment. An antiapoptotic gene (Birc2), I kappa B-xi, and protective gene (HO-I) were also upregulated. Other upregulated genes included oncogenes (lipocalin2, Bcl3, and CCAAT/enhancer binding protein delta), Calgranulin B, DEXRAS1, insulin-like growth factor binding protein-1, inhibin beta-B-subunit gene, IgG Fc receptor, and FK 506 binding protein 5. We also observed downregulation of the genes Amphiphsin, Jagged 1, Pace 4, Slc15a2, Kcnn2, and gap junction membrane channel protein alpha 5 only in kidneys from BD donors. Conclusions: This is the first demonstration of global gene expression analysis using the rat brain-death isograft model. These results provide new insights for the detection of novel target genes for treatment and prognosis of grafts from brain-dead and extended marginal donors.

There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.