Enhancement of autophagy is a potential modality for tumors refractory to radiotherapy

Radiotherapy is a well-established treatment for cancer. However, the existence of radioresistant cells is one of the major obstacles in radiotherapy. In order to understand the mechanism of cellular radioresistance and develop more effective radiotherapy, we have established clinically relevant radioresistant (CRR) cell lines, which continue to proliferate under daily exposure to 2 Gray (Gy) of X-rays for >30 days. X-ray irradiation significantly induced autophagic cells in parental cells, which was exiguous in CRR cells, suggesting that autophagic cell death is involved in cellular radiosensitivity. An autophagy inducer, rapamycin sensitized CRR cells to the level of parental cells and suppressed cell growth. An autophagy inhibitor, 3-methyladenine induced radioresistance of parental cells. Furthermore, inhibition of autophagy by knockdown of Beclin-1 made parental cells radioresistant to acute radiation. These suggest that the suppression of autophagic cell death but not apoptosis is mainly involved in cellular radioresistance. Therefore, the enhancement of autophagy may have a considerable impact on the treatment of radioresistant tumor.     

Isolation of a Spore Germination Inhibitor from a Cellular Slime Mold Dictyostelium discoideum

The functional properties of gluten obtained by treating with chymotrypsin at alkali pH were investigated. The gluten was treated by chymotrypsin at pH 10.0 and 20°C, and was found to be deamidated to a state that was scarcely subject to proteolysis by chymotrypsin. The degree of deamidation of the gluten reached about 25% by this treatment for 2 hr. The functional properties of the gluten thus obtained were investigated in regard to deamidation. The enzymatically deamidated gluten greatly improved such functional properties as solubility and emulsifying ability. In particular, the solubility of the treated gluten was remarkably high in the pH range of 5 to 8, in which native gluten is insoluble. It was apparent that the improvement in functional properties of gluten was mainly due to the deamidation induced by treating with chymotrypsin at pH 10.0 and 20°C.     

Effect of traditional floor sitting on postural control after standing

Seiza is a Japanese traditional floor sitting style, sitting down with both legs set at about a 180 degree angle and both femurs on both lower legs. We examined the influence of the somatic dysesthesia and decrease in voluntary toe flexion strength (VTF) induced by Seiza on the center of pressure (COP) sway. Fifteen adults participated in this experiment. COP Sway was measured immediately after a chair resting (pre-test), when a plantar dysethesia occurred (post-test A), and when a decrease (under 30% of maximal voluntary contraction (MVC)) in the VTF set in (post-test B). Tissue oxygenation kinetics in the soleus muscle and plantar somatosensory thresholds (ST) were measured just before each COP test and during Seiza. From starting Seiza, oxygenated hemoglobin/myoglobin decreased markedly and reached a plateau within about 6 min. ST abruptly increased at about 19 min from starting Seiza. VTF decreased to less than 30% MVC in 33% of the participants after 10 min from the acute increase in ST, and in 100% after 20 min. When sustaining Seiza for 19 min, ST rose and sway velocity and antero-posterior sway increased. With continued Seiza, VTF decreased to below 30% MVC at 10 - 20 min, and the above stated body sway further markedly increase.     

Intestinal and renal adaptation to a low-Pi diet of type II NaPi cotransporters in vitamin D receptor- and 1alphaOHase-deficient mice

Intake of a low-phosphate diet stimulates transepithelial transport of P_i in small intestine as well as in renal proximal tubules. In both organs, this is paralleled by a change in the abundance of the apically localized NaP_i cotransporters NaP_i type IIa (NaP_i-IIa) and NaP_i type IIb (NaP_i-IIb), respectively. Low-P_i diet, via stimulation of the activity of the renal 25-hydroxyvitamin-D₃-1-hydroxylase (1OHase), leads to an increase in the level of 1,25-dihydroxy-vitamin D₃ [1,25(OH)₂D]. Regulation of the intestinal absorption of P_i and the abundance of NaP_i-IIb by 1,25(OH)₂D has been supposed to involve the vitamin D receptor (VDR). In this study, we investigated the adaptation to a low-P_i diet of NaP_i-IIb in small intestine as well as NaP_i-IIa in kidneys of either VDR- or 1OHase-deficient mice. In both mouse models, upregulation by a low-P_i diet of the NaP_i cotransporters NaP_i-IIa and NaP_i-IIb was normal, i.e., similar to that observed in the wild types. Also, in small intestines of VDR- and 1OHase-deficient mice, the same changes in NaP_i-IIb mRNA found in wild-type mice were observed. On the basis of the results, we conclude that the regulation of NaP_i cotransport in small intestine (via NaP_i-IIb) and kidney (via NaP_i-IIa) by low dietary intake of P_i cannot be explained by the 1,25(OH)₂D-VDR axis. NaP_i type IIb; vitamin D₃     

Proteolytic degradation of glutamate decarboxylase mediates disinhibition of hippocampal CA3 pyramidal cells in cathepsin D-deficient mice

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.     

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the brain of a teleost, Uranoscopus japonicus: immunohistochemical relationship between PACAP and adenohypophysial hormones

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) could play a role in stimulating pituitary hormone release in fish brain. In this study, we used immunochemical techniques to examine the histological and quantitative distribution of PACAP in the central nervous system (CNS) of a teleost, the stargazer, Uranoscopus japonicus. In addition, high performance liquid chromatographic (HPLC) analysis was performed to characterize the form of PACAP present, while the relationship between PACAP and adenohypophysial hormones was also determined immunohistochemically. PACAP-like immunoreactive (LI) neuronal cell bodies and fibers were found not only in the hypothalamo-pituitary region but also in the midbrain and hindbrain regions. PACAP-LI fibers were identified in the neurohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. The concentration of immunoreactive PACAP in whole brain tissue was approximately 300 pmol/g wet weight. The average concentrations of immunoreactive PACAP in regions of the telencephalon, diencephalon, tectum, cerebellum, and rhombencephalon were 217.53, 510.26, 83.30, 148.64, and 364.62 pmol/g, respectively. In reverse-phase HPLC experiments, the predominant form of immunoreactive PACAP eluted closely with synthetic stargazer PACAP38, while PACAP27-like immunoreactivity was negligible. These results suggest that PACAP38 is the predominant PACAP form in the stargazer CNS, and that PACAP acts not only as a hypophysiotropic factor for adenohypophysial hormone release but also as a neurotransmitter and neuromodulator in the CNS.     

Effect of Japanese sitting style (seiza) on the center of foot pressure after standing

Seiza is one of the most commonly used sitting postures in various enrichment lessons of Japanese origin. It is reported that Seiza with large knee flexion produces harmful effects on the cartilage of knee joints and hemodynamics of the lower legs. This study aimed at examining the influence of Seiza on tissue oxygenation kinetics of the lower limbs, plantar somatic and cutaneous sensation, and the center of foot pressure (COP) sway using 10 young adults. COP sway was measured for 1 min just after sitting on a chair for 10 min (pre-test), after 30-min Seiza (post-test 1), and 5 min after Seiza (post-test 2). To evaluate the COP sway, we used 4 body sway factors; unit time sway factor (F1), front-back sway factor (F2), left-right sway factor (F3) and high frequency band power spectrum factor (F4). Physiological parameters (i.e., tissue oxygenation kinetics in the lower legs and sensation on the sole) were measured during 30-min Seiza (continuously on tissue oxygenation, and at 1 min intervals on sensation), and for 1 min just before each COP test (pre-test, post-test 1 and 2).Oxygenated hemoglobin/myoglobin (Hb/Mb) concentration decreased markedly and deoxygenated Hb/Mb concentration increased markedly, resulting in reaching a plateau state at around 7 min. Tissue Hb/Mb index changed little during Seiza. Proprioceptive perception thresholds increased rapidly about 17 min after Starting Seiza. Means of 3 COP sway factors of F1, F2 and F4 were significantly higher in post-test 1 than in pre-test and post-test 2. In conclusion, a marked decrease in tissue oxygen concentration of the lower legs within 4-5 min, and an increase of proprioceptive perception thresholds in the sole at about 17 min are induced by Seiza. Although wiggle and quick body sway in the antero-posterior axis increases markedly in an upright posture just after maintaining Seiza for 30 min, sway recovers after sitting on a chair for 5 min.