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Time perception varies across different sensory modalities, with previous research indicating that visual flicker can dilate perceived duration while auditory flutter can sometimes compress it. This study aimed to explore duration perception in the relatively understudied tactile modality and compare it with the visual modality. In Experiment 1A, we investigated the effect of temporal modulation on perceived duration using 1-s tactile flutter and visual flicker stimuli, either stationary or modulated at 6.5 Hz, 12.5 Hz, or 24.5 Hz. Participants reported which of two sequentially presented stimuli appeared longer in duration. Results demonstrated that both tactile flutter and visual flicker induced significant time distortions, with higher frequencies leading to greater duration dilation. Notably, the magnitudes of time distortion were significantly correlated between the two modalities. Experiment 1B assessed the roles of sensitivity and subjective saliency in these time distortions. Contrary to previous findings, saliency did not correlate with the observed time distortions in either modality, and sensitivity did not correlate with time distortions in the visual modality. These results suggest that, while tactile duration perception exhibits similarities to visual duration perception, the lack of correlation with saliency challenges the idea that time distortion is driven by subjective saliency. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a progressive neuropsychiatric disease affecting many elderly people and is characterized by progressive cognitive impairment of memory, visuospatial, and executive functions. As the elderly population is growing, the number of AD patients is increasing considerably. There is currently growing interest in determining AD's cognitive dysfunction markers. We used exact low-resolution-brain-electromagnetic-tomography independent-component-analysis (eLORETA-ICA) to assess activities of five electroencephalography resting-state-networks (EEG-RSNs) in 90 drug-free AD patients and 11 drug-free patients with mild-cognitive-impairment due to AD (ADMCI). Compared to 147 healthy subjects, the AD/ADMCI patients showed significantly decreased activities in the memory network and occipital alpha activity, where the age difference between the AD/ADMCI and healthy groups was corrected by linear regression analysis. Furthermore, the age-corrected EEG-RSN activities showed correlations with cognitive function test scores in AD/ADMCI. In particular, decreased memory network activity showed correlations with worse total cognitive scores for both Mini-Mental-State-Examination (MMSE) and Alzheimer's Disease-Assessment-Scale-cognitive-component-Japanese version (ADAS-J cog) including worse sub-scores for orientation, registration, repetition, word recognition and ideational praxis. Our results indicate that AD affects specific EEG-RSNs and deteriorated network activity causes symptoms. Overall, eLORETA-ICA is a useful, non-invasive tool for assessing EEG-functional-network activities and provides better understanding of the neurophysiological mechanisms underlying the disease. [ABSTRACT FROM AUTHOR]

Secondary necrotic cells, which are generated if apoptotic cells are incompletely cleared, induce severe inflammatory responses involving MIP-2 production and subsequent neutrophil infiltration. Recently, we showed that the phagocytic capacity of peritoneal resident macrophages from wild type (WT) aged mice as well as SMP30 −/− mice fed a VC-limited diet as to secondary necrotic cells was reduced as compared with that in young mice, and that the inflammatory responses induced were stronger than those in young mice, presumably because of the delay in removal of secondary necrotic cells in aged mice. In this study, we investigated why MIP-2 production was increased in aged mice upon injection of secondary necrotic cells and why the phagocytic capacity of peritoneal resident macrophages from aged mice was reduced. When cocultured with secondary necrotic cells, the peritoneal resident macrophages from both types of aged mice significantly produced MIP-2 even in the absence of IFN-γ, whereas MIP-2 production by macrophages from WT young mice required IFN-γ. The peritoneal resident macrophages from both types of aged mice expressed CD40, a M1 macrophage marker, as in the case of M1 macrophages, which were obtained by treatment of macrophages from WT young mice with IFN-γ and LPS. Furthermore, M1 macrophages exhibited less phagocytic capacity as to secondary necrotic cells than non-treated macrophages. These results suggest that the phenotype of peritoneal resident macrophages is skewed toward M1-like in aged mice and that such skewing toward M1-like is involved in enhancement of inflammatory responses induced by secondary necrotic neutrophils in aged mice. [ABSTRACT FROM AUTHOR]

Aim Secondary necrotic cells generated in vivo induce inflammatory responses; for example, the production of macrophage inflammatory protein-2 ( MIP-2) and subsequent infiltration of neutrophils. The aim of the present study was to elucidate the effect of aging on the phagocytosis of secondary necrotic cells and the inflammatory responses by using either wild-type ( WT) young mice, WT aged mice or senescence-accelerated mice ( SMP30−/− mice). Methods The phagocytosis of secondary necrotic neutrophils with resident macrophage from either WT young mice, WT aged mice or SMP30−/− mice was examined by coculturing macrophages with secondary necrotic neutrophils in vitro. To investigate the inflammatory response induced by secondary necrotic cells, time-dependent infiltration of neutrophils and production of MIP-2 were determined in the peritoneal cavity on the injection of secondary necrotic cells. Results The phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30−/− mice was significantly reduced as compared with that by macrophages from WT young mice. On peritoneal injection of secondary necrotic cells, the peak time of neutrophil infiltration was earlier in SMP30−/− mice than in WT young mice. The number of neutrophils in SMP30−/− mice at the peak time was also greater than that in WT young mice. Conclusions Our findings showed that the phagocytosis of secondary necrotic cells was attenuated in aged mice and SMP30−/− mice, and that the MIP-2 production was enhanced and subsequently neutrophil infiltration was exaggerated on peritoneal injection of secondary necrotic cells into those mice. Geriatr Gerontol Int 2016; 16: 135-142. [ABSTRACT FROM AUTHOR]

Abstract: Annexin A1 (ANXA1) is a well-known anti-inflammatory protein that is expressed on the surface of apoptotic cells. Annexin A4 (ANXA4) is also recruited from the nucleus to the cytoplasm in apoptotic cells, although it is not known whether or not ANXA4 is expressed on the surface of apoptotic cells. In this study, we obtained rabbit anti-human ANXA1 and ANXA4 antibodies, and then examined whether or not ANXA1 and ANXA4 are expressed on the surface of early and late human apoptotic cells. ANXA1 and, to a lesser extent, ANXA4 were detected on late but not early apoptotic HeLa cells, whereas ANXA1 and a small amount of ANXA4 were detected on both early and late apoptotic human neutrophils. We then examined the effects of the anti-human ANXA1 and ANXA4 antibodies on the mouse or human macrophage response to human apoptotic cells. Upon coculturing of mouse or human macrophages with late apoptotic human neutrophils, anti-human ANXA1 antibodies and, to a lesser extent, anti-human ANXA4 antibodies increased MIP-2 or IL-8 production significantly, suggesting that ANXA1 and ANXA4 suppress MIP-2 or IL-8 production by macrophages in response to late apoptotic human neutrophils. [Copyright &y& Elsevier]

The matrix metalloproteinase (MMP) family (~25 members in mammals) has been implicated in extracellular matrix remodeling associated with embryonic development, cancer formation and progression, and various other physiological and pathological events. Inactivating mutations in individual matrix metalloproteinase genes in mice described so far, however, are nonlethal at least up to the first few weeks after birth, suggesting functional redundancy among MMP family members. Here, we report that mice lacking two MMPs, MMP-2 (nonmembrane type) and MT1-MMP (membrane type), die immediately after birth with respiratory failure, abnormal blood vessels, and immature muscle fibers reminiscent of central core disease. In the absence of MMP-2 and MT1-MMP, myoblast fusion in vitro is also significantly retarded. These findings suggest functional overlap in mice between the two MMPs with distinct molecular natures.Oncogene (2004) 23, 5041-5048. doi:10.1038/sj.onc.1207688 Published online 5 April 2004 [ABSTRACT FROM AUTHOR]

Background: The increased use of esophagogastroduodenoscopy has revealed the esophagus to be a common site of occurrence of granular cell tumors (GCT). To clarify the endosonographic features of esophageal GCT, we compared endosonographic findings and histopathological analysis of resected specimens of esophageal GCT and submucosal leiomyomas. Methods: We enrolled a total number of 18 consecutive patients with esophageal submucosal tumors. They were preoperatively examined using 15 MHz or 20 MHz probe endoscopic ultrasonography. The endosonographic findings were retrospectively compared with hisopathological findings of the resected specimens. Results: Five GCT and 13 submucosal leiomyomas were diagnosed histologically as well as endosonographically. Pathological analysis revealed that the dispropotional mixture of tumor cells, fibrosis and muscularis mucosa within the GCT would be endosonographically visualized as a heterogeneous hypoechoic mass. Moreover, the laterally scattered tumor cells, together with adjacent thickened muscularis mucosa, are reflected by the unclear margin of the tumor by endoscopic ultrasonography. In contrast, in the submucosal leiomyomas, tumor cells and fibrosis were uniformly mixed and the margin of the tumor was clearly demarcated. These histological findings would be endosonographically recognized as a homogeneously hypoechoic mass with a clearly demarcated border. Conclusion: A high-frequency probe endoscopic ultrasonography is useful for differentiating esophageal GCT from submucosal leiomyomas. Considering the histological characteristics, esophageal GCT should be resected with adequate tumor-free margin when endoscopic treatment is applied. [ABSTRACT FROM AUTHOR]

Examines the importance of matrix metalloproteinases (MMP) for proper extracellular matrix remodelling. Inhibition of tumor invasion and metastasis; Involvement of MMP in cancer progression; Detection of defects in collagen fibrils.

Recent studies have suggested that wild-type p53 blocks cell cycle progression near the G1-S boundary and is involved in both differentiation and apoptosis in many types of cells including cancer cells, p53 expression is enhanced upon DNA-damaging apoptotic stimuli while Fas/Apo-1, a member of the tumor necrosis factor receptor family expressed on cell surface, transduces a signal for apoptosis upon specific ligand or antibody engagement. We demonstrated that stable transfection of the wild-type p53 gene under the control of CMV promoter induced differentiation and apoptosis under restricted conditions in cancer cells, and often caused sensitization of p53-transfected cells to Fae/Apo-1 signal. To investigate the interaction between two major apoptotic pathways involving p53 and Fas/Apo-1 we have established a system that allows to induce wild-type p53 overexpression and apoptosis in cancer cells upon treatment with anti-Fas antibody. The system also allows to investigate other factors interacting with p53 and Fas/Apo-1, and should provide a clue to understanding the biological and biochemical aspects of apoptosis. [ABSTRACT FROM AUTHOR]

Hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) is a candidate of radiosensitizers which enables both anti-tumour and anti-metastasis effects in X-ray therapy. The curative effects under such 4-MU administration have been investigated in vitro; however, the radiosensitizing mechanisms remain unclear. Here, we investigated the radiosensitizing effects under 4-MU treatment from cell experiments and model estimations. We generated experimental surviving fractions of human fibrosarcoma cells (HT1080) after 4-MU treatment combined with X-ray irradiation. Meanwhilst, we also modelled the pharmacological effects of 4-MU treatment and theoretically analyzed the synergetic effects between 4-MU treatment and X-ray irradiation. The results show that the enhancement of cell killing by 4-MU treatment is the greatest in the intermediate dose range of around 4 Gy, which can be reproduced by considering intercellular communication (so called non-targeted effects) through the model analysis. As supposed to be the involvement of intercellular communication in radiosensitization, the oxidative stress level associated with reactive oxygen species (ROS), which leads to DNA damage induction, is significantly higher by the combination of 4-MU treatment and irradiation than only by X-ray irradiation, and the radiosensitization by 4-MU can be suppressed by the ROS inhibitors. These findings suggest that the synergetic effects between 4-MU treatment and irradiation are predominantly attributed to intercellular communication and provide more efficient tumour control than conventional X-ray therapy. [ABSTRACT FROM AUTHOR]

The vagus nerve is the only pathway for transmitting parasympathetic signals between the brain and thoracoabdominal organs, thereby exhibiting anti-inflammatory functions through the cholinergic anti-inflammatory pathway. Despite often being resected during lymph node dissection in upper gastrointestinal cancer surgery, the impact of vagotomy on postoperative outcomes in gastric cancer patients remains unclear. Sub-diaphragmatic vagotomy was performed on C57BL/6 mice. Three weeks later, syngeneic murine gastric cancer cell line YTN16P was injected into the peritoneal cavity, and the number of peritoneal metastases (PM) on the mesentery and omentum compared with control mice. The phenotypes of immune cells in peritoneal lavage and omental milky spots one day after tumor inoculation were analyzed using flow cytometry and immunohistochemistry. Intraperitoneal transfer of 3 × 105 YTN16P significantly increased the number of metastatic nodules on the mesentery in the vagotomy group compared to the control group. The omental metastasis grade was also significantly higher in the vagotomy group. Phenotypic analysis of immune cells in peritoneal lavage did not reveal significant differences after vagotomy. However, vagotomized mice exhibited a notable increase in milky spot area, with a higher presence of cytokeratin(+) tumor cells, F4/80(+) macrophages, and CD3(+) T cells. Vagus nerve signaling appears to regulate the immune response dynamics within milky spots against disseminated tumor cells and inhibits the development of PM. Preserving the vagus nerve may offer advantages in advanced gastric cancer surgery to reduce peritoneal recurrence. [ABSTRACT FROM AUTHOR]

Background: We aimed to validate the Clinical Dementia Rating (CDR®) dementia staging instrument plus the National Alzheimer's Coordinating Centre Behaviour and Language Domains (CDR® plus NACC FTLD) for use in clinical settings in Japan and in the Japanese language. Methods: This prospective observational study enrolled 29 patients with frontotemporal dementia (FTD) and 21 patients with Alzheimer's disease (AD) dementia from the Departments of Psychiatry at Osaka University Hospital and Asakayama General Hospital and the Brain Function Centre at Nippon Life Hospital. CDR® plus NACC FTLD, CDR®, Mini‐Mental State Examination (MMSE), Western Aphasia Battery (WAB), Neuropsychiatric Inventory‐plus (NPI‐plus), Stereotypy Rating Inventory (SRI), and frontal behavioural symptom scores obtained from items of NPI‐plus and SRI, were conducted to assess inter‐ and intra‐rater reliability, validity, and responsiveness. We performed receiver operating characteristic (ROC) curve analysis to evaluate the discriminating power of the Behaviour/Comportment/Personality (BEHAV) and Language (LANG) domains of the CDR® plus NACC FTLD and the MEMORY domain of the CDR® in patients AD dementia and FTD. Results: The CDR® plus NACC FTLD showed good inter‐ and intra‐rater reliabilities. In patients with FTD, the BEHAV domain of the CDR® plus NACC FTLD was significantly correlated with all clinical measures except for the SRI total score, while the LANG domain of the CDR® plus NACC FTLD was significantly correlated with the MMSE and the WAB‐Aphasia quotient. In addition, the CDR® plus NACC FTLD sum of boxes significantly changed after 6 months and after 1 year. ROC curve analysis showed that the BEHAV and LANG domains of the CDR® plus NACC FTLD distinguished between patients with AD dementia and FTD better than the MEMORY domain of the CDR®. Conclusions: This study validated the Japanese version of the CDR® plus NACC FTLD with good reliability, validity, and responsiveness. [ABSTRACT FROM AUTHOR]

Investigates the clonal origin of multifocal urothelial tumors. Analysis of the p53 tumor-suppressor gene in cases with bladder tumors developing after treatment for a renal of ureteral tumor; Detection of identical p53 gene mutations in all primary and recurrent tumors; Finding that heterotopic recurrence by intraluminal seeding from the original tumor is common in urothelial cancer.

An alternatively splicing of the rat erythropoietin receptor (EpoR) gene was identified in normal and erythroleukemia cells by the reverse transcription PCR method. Insertion of a 105 bp fragment at the region corresponding to the extracellular domain of rat EpoR was found. The insert sequence, which encodes additional 21 amino acids, is similar to that previously found in the mouse EpoR gene, however, has an additional 27 bp direct repeat. Due to the presence of a stop codon in the insert, the alternative transcript is translated to a truncated and soluble form of EpoR which is preferentially expressed in liver, spleen, kidney, heart, and bone marrow cells as well as cultured erythroleukemia cells. These findings suggest that alternative splicing of the EpoR gene may play an important role in proliferation and differentiation of rat erythroid cells. [ABSTRACT FROM AUTHOR]

The membrane-anchored MMP-regulator RECK is down regulated in many solid tumors; the extent of RECK down regulation correlates with poor prognosis. Forced expression of RECK in tumor cells results in suppression of angiogenesis, invasion, and metastasis. Studies on the roles and the mechanisms of regulation of the RECK gene during normal development may therefore yield important insights into how the malignant behaviors of tumor cells arise and how they can be controlled. Our previous studies indicate that mice lacking RECK die around E10.5 with reduced tissue integrity. In the present study, we have found that in later stage wild-type embryos, RECK is abundantly expressed in skeletal muscles, especially in the areas where the myoblast differentiation factor MRF4 is expressed. Consistent with this finding, the RECK-promoter is activated by MRF4 in cultured cells. In contrast, a myoblast determination factor MyoD suppresses the RECK-promoter. Myoblastic cells lacking RECK expression give rise to myotubes at higher efficiency than the cells expressing RECK, indicating that RECK suppresses myotube formation. These findings suggest that MyoD down regulates RECK to facilitate myotube formation, whereas MRF4 up regulates RECK to promote other aspects of myogenesis that require extracellular matrix integrity.Oncogene (2005) 24, 5850–5857. doi:10.1038/sj.onc.1208733; published online 4 July 2005 [ABSTRACT FROM AUTHOR]

Previous studies indicate that immunostimulatory DNA-based injectable hydrogels harboring unmethylated cytosine-phosphate-guanine (CpG) dinucleotides meet the requirements of an effective antigen delivery system, including safety, biodegradability, ease of administration, and stimulation of the innate immune system. However, rapid release of the model antigen ovalbumin (OVA) from the hydrogel limits its potential. Here, the aim is to achieve sustained OVA release from a DNA hydrogel through cationization of the antigen. Ethylenediamine (ED)-conjugated cationized OVA (ED-OVA), but not OVA, forms a complex with hexapod-like structured DNA, a component of the DNA hydrogel. The release of ED-OVA from the hydrogel is significantly slower than that of OVA. ED-OVA mixed with CpG DNA hydrogel efficiently binds to mouse dendritic DC2.4 cells and results in high antigen presentation. Intratumoral injections of ED-OVA/CpG DNA hydrogel significantly delays tumor growth of OVA-expressing EG7-OVA cells in mice. Then, a cationic OVA peptide antigen (R8-L2-pepI) consisting of an OVA MHC class I epitope, octaarginine, and a linker is designed. Intratumoral injections of R8-L2-pepI/CpG DNA hydrogel eradicate tumors in five out of six mice. Thus, it is concluded that a vaccine consisting of immunostimulatory CpG DNA hydrogel and cationized antigens can be effective for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract: DNA nanotechnology-based nanosystems and macrosystems have attracted much attention in the biomedical research field. The nature of DNA endows these systems with biodegradable, biocompatible, and immunomodulatory properties. Here, we present an injectable hydrogel system that consists only of chemically synthesized short DNA strands, water, and salts. Several preparations of polypod-like structured DNA, or polypodna, were designed, including tri-, tetra-, penta- and hexapodna, as the building blocks of self-gelling DNA hydrogel. Under physiological conditions, properly designed polypodna preparations formed a hydrogel. The analysis of the modulus data of the hydrogel consisting of two sets of hexapodna preparations showed that this injectable hydrogel was reorganized at a time scale of 0.25s. Then, DNA hydrogel containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides was used to stimulate innate immunity through Toll-like receptor 9, the receptor for CpG DNA. Gel formation significantly increased the activity of immunostimulatory CpG DNA, retarded the clearance after intradermal injection into mice, and increased the immune responses to ovalbumin (OVA) incorporated into the hydrogel as a model antigen. OVA/CpG DNA hydrogel induced much less local or systemic adverse reactions than OVA injected with complete Freund's adjuvant or alum. GpC DNA hydrogel containing no CpG sequences was less effective, indicating the importance of immunomodulation by CpG DNA hydrogel. Thus, we have created an efficient system for sustained delivery of antigens or other bioactive compounds. [Copyright &y& Elsevier]

Background and Aim: Application of acetic acid topically to the mucosal or serosal side of the stomach has been well used to create a chronic gastric ulcer model. The aim of the present study was to apply it as a new cytoreductive approach in a mouse model of gastric cancer. Methods: A total of 43 genetically engineered mice, the so-called (INS-GAS) mice that develop spontaneously gastric cancer at 10-14 months of age, were included. Acetic acid-induced ulcer method was applied to mice under isofluran anesthesia. The ulcer at the cancer side was made by exposing either the anterior serosal or posterior mucosal side of gastric wall to 0.1 mL of 60% or 100% acetic acid for 30 or 60 s with a cylindrical metal mold (4 mm ID). Route to the serosal side was intra-abdominal and one to the mucosal side was through a small hole made in the forestomach. The opposite side of gastric wall (no treatment with acetic acid) was used as the corresponding control. After the mice were sacrificed, the stomachs were collected 1, 3, 6 h or 1, 3 and 7 days, postoperatively, and evaluated by visual inspection and histology. Results: Gastric cancer was found in both the anterior and posterior walls of the corpus in all 43 mice. Intraluminal pH value was between 11 and 13. Severe necrosis in the cancer was observed in the side exposing to acetic acid, but not in the control side, shortly after the treatment (i.e. within 30 or 60 min). The muscularis mucosa and muscle layers were less damaged, regardless of the side of the treatment. Ulcer formation in the cancer took place 1, 3 or 7 days later. The ulcer depth was sometimes at the muscularis mucosa and muscle layers. At 3 and 7 days, regeneration of epithelial cells was clearly observed in the ulcer margin in the stomach of mice. Conclusions: Topical application of acetic acid either from mucosal or serosal surface promptly caused the necrosis of tumor, suggesting the potential approach of this simple and reliable method as a cytoreductive treatment of gastric cancer in patients through endoscopy or laparoscopy. [ABSTRACT FROM AUTHOR]

Background: Developmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear. Results: We found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos. Conclusions: Our findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyornic tissues. [ABSTRACT FROM AUTHOR]

Nerve apposition on nicotinic acetylcholine receptor clusters and invagination of the post-synaptic membrane (i.e. secondary fold formation) occur by embryonic day 18.5 at the neuromuscular junctions (NMJs) in mouse skeletal muscles. Finding the molecules expressed at the NMJ at this stage of development may help elucidating how the strong linkage between a nerve terminal and a muscle fiber is established. Immunohistochemical analyses indicated that the membrane-anchored matrix metalloproteinase regulator RECK was enriched at the NMJ in adult skeletal muscles. Confocal and electron microscopy revealed the localization of RECK immunoreactivity in secondary folds and subsynaptic intracellular compartments in muscles. Time course studies indicated that RECK immunoreactivity becomes associated with the NMJ in the diaphragm at around embryonic day 18.5 and thereafter. These findings, together with known properties of RECK, support the hypothesis that RECK participates in NMJ formation and/or maintenance, possibly by protecting extracellular components, such as synaptic basal laminae, from proteolytic degradation. [ABSTRACT FROM AUTHOR]

We report that during cortical development in the mouse embryo, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) critically regulates Notch signaling by antagonizing the ectodomain shedding of Notch ligands, which is mediated by a disintegrin and metalloproteinase domain 10 (ADAM10). In the embryonic brain, RECK is specifically expressed in Nestin-positive neural precursor cells (NPCs). Reck-deficient NPCs undergo precocious differentiation that is associated with downregulated Nestin expression, impaired Notch signaling and defective self-renewal. These phenotypes were substantially rescued either by enhancing Notch signaling or by suppressing endogenous ADAM10 activity. Consequently, we found that RECK regulates the ectodomain shedding of Notch ligands by directly inhibiting the proteolytic activity of ADAM10. This mechanism appeared to be essential for Notch ligands to properly induce Notch signaling in neighboring cells. These findings indicate that RECK is a physiological inhibitor of ADAM10, an upstream regulator of Notch signaling and a critical modulator of brain development. [ABSTRACT FROM AUTHOR]

Hatano E, Tanaka A, Kanazawa A, Tsuyuki S, Tsunekawa S, Iwata S, Takahashi R, Chance B, Yamaoka Y. Inhibition of tumor necrosis factor-induced apoptosis in transgenic mouse liver expressing creatine kinase. Liver International 2004: 24: 384–393. © Blackwell Munksgaard 2004 The mitochondrion acts as a pivotal decision center in many types of apoptotic responses. To clarify the effects of the enhanced mitochondrial function on tumor necrosis factorα (TNFα)-induced apoptosis, we studied hepatic injuries in transgenic mice whose livers express creatine kinase (CK). Mice fed a diet containing 10% creatine, came to accumulate phosphocreatine and to enhance hepatic ATP levels and mitochondrial oxidative phosphorylation activities. TNFα-mediated hepatic apoptosis in normally fed and Cr-feeding CK transgenic mice were assessed. TNFα and actinomycin D cause severe liver failure in normally fed transgenic mice, and in the wild-type mice. In contrast, no significant elevations in transaminase levels after injection were observed in Cr feeding transgenic mice. The disruption of the mitochondrial transmembrane potential at 2 h after TNFα injection, prior to ATP depletion, activation of caspase 3 like protease, and DNA fragmentation at 4–6 h after injection, were observed in normally fed transgenic mice. These were fully suppressed in Cr feeding transgenic mice. However, anti-Fas antibody-induced apoptosis was not inhibited in both groups. The results indicate that TNFα-induced apoptosis was inhibited in CK transgenic mice livers by maintaining mitochondrial function. [ABSTRACT FROM AUTHOR]

p16, cyclin D1 and retinoblastoma protein (pRB) regulate G[sub 1] to S transition and are commonly targeted in various cancers. However, few studies have simultaneously examined all components of the p16/cyclin D1/pRB pathway (RB pathway) in hepatocellular carcinoma (HCC). To clarify the role of the disruption of the RB pathway in HCC, we analyzed p16, pRB and cyclin D1 in 47 HCCs. Inactivation of p16 was detected in 30 of 47 HCCs (64%) by Western blot analysis and significantly correlated with hypermethylation of the promoter of this gene. pRB expression was found to be absent in 13 of 47 HCCs (28%) by immunohistochemistry. We found that 38 of 47 HCCs (81%) contained at least one inactivation in either pRB or p16. Furthermore, there was a significant inverse correlation between p16 and pRB inactivation (p = 0.041). Overexpression of cyclin D1 was detected in 5 of 47 HCCs (11%) by immunohistochemistry. The cases with cyclin D1 overexpression exhibited an advanced clinicopathological appearance and also contained inactivation of pRB and/or p16. These findings suggest that inactivation of pRB and/or p16 is a major event in human hepatocarcinogenesis, while cyclin D1 overexpression may confer additional growth advantages to the tumor in addition to pRB and/or p16 inactivation in HCC.Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Conditions of preserving DNA, RNA and protein in pathological specimens are of great importance as degradation of such macromolecules would critically affect results of molecular biological analysis. The feasibility of freeze-drying as a means of preserving pathological tissue samples for molecular analysis has previously been shown. In the present study, further tests on long-term storage conditions and analyses of freeze-dried samples by polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR, western blotting and immunohistochemistry are reported. Rat chromosomal DNA of freeze-dried samples stored for 4 years showed slight degradation while RNA degradation was more prominently seen at an earlier stage of storage. However, these 4 year DNA and RNA samples were still able to serve as a template for some PCR and RT-PCR analyses, respectively. Overexpression of c-erbB-2 and p53 protein was demonstrated by western blotting and immunohistochemical staining using freeze-dried human breast cancer tissues. Although macromolecules in freeze-dried samples degrade to some extent during the preservation period, they should still be of value for certain molecular biological analyses and morphological examination; hence, providing more convenient and inexpensive ways of pathological tissue storage. [ABSTRACT FROM AUTHOR]

L5176 --> Osmolytes are small organic molecules that demonstrate the ability to shift protein unfolding equilibria when present as solution components, and provide proteins with protection from denaturation against thermal stress. While many subclasses of osmolytes are known, it is unclear whether there is an intrinsic stabilization hierarchy among the molecules that applies to all proteins. Additionally, it is uncertain what physicochemical properties of osmolytes confer their ability to act as stabilizers or destabilizers in solution. Our investigation utilizes differential scanning fluorimetry (DSF) to measure protein stability in two structure‐based surveys: 1) determining whether there is a stabilization hierarchy among the three main chemical classes of osmolytes with respect to the model proteins, human C‐reactive protein (CRP) and tumor necrosis factor alpha (TNFα), and 2) investigating the structural moiety requirements for amino acid osmolytes in the stabilization of CRP and myoglobin. The former part of our investigation reveals that ectoine is the most effective stabilizer of CRP, yet is a destabilizer when acting on TNFα, suggesting that there is no one osmolyte, or class of osmolytes, that confers the greatest stability across multiple proteins. The latter part of our investigation reveals that myoglobin is stabilized only by zwitterionic amino acids, as removal of either the carboxylate or amino moiety from the amino acid abrogates all thermal stabilizing effects. In contrast, in the case of CRP the carboxylate proved to be the dominant stabilizing group, as compounds lacking the amino group conferred slightly greater thermal stability than the complete amino acid osmolyte. Collectively, our results affirm that osmolyte effects on protein thermal stability are highly protein‐specific. In addition, our data suggests that contemporary models to explain the osmolyte effect may not apply to all protein systems. These results may have significant implications for the development of chemistries aiming to stabilize diverse protein targets in biospecimens for diagnostic applications. [ABSTRACT FROM AUTHOR]

L5059 --> Multiple antibiotic resistance regulator (MarR) proteins are widely found in prokaryotes and archaea. Most characterized MarR homologs function as transcriptional repressors of genes involved in metabolic and stress response pathways. MarR proteins regulate transcription through sequence‐specific DNA binding interactions and many respond allosterically to small molecule ligands or metal ions. In this report, we describe the DNA and ligand binding properties of PaeR (putative antibiotic efflux regulator), a previously uncharacterized MarR homolog from the pathogen, Clostridium difficile. Biophysical characterization of PaeR indicates that it exists predominantly as a homodimer with a thermal unfolding temperature of 59.3°C. Electrophoretic mobility shift assays (EMSAs) reveal PaeR to be a sequence‐specific DNA binding protein that associates with DNA as a homodimer with very high binding affinity (Kd = 3.2 ± 0.6 nM) at a pseudo‐palindromic site in its promoter region. Interestingly, our data indicates that PaeR binds to at least one additional site in its promoter/operator region with positive binding cooperativity. Initial screens to identify potential effectors of PaeR binding to DNA indicate that this protein is responsive to low concentrations of copper ions. Homology modeling of the PaeR homodimer suggests a possible mechanism for copper‐induced effects on DNA binding. These data, in conjunction with the genetic context of the paeR gene, suggest a possible role of PaeR in regulating antibiotic resistance in C. difficile. [ABSTRACT FROM AUTHOR]

Recent studies have reported that apoptosis may be induced by reperfusion injury following ischemia in cardiomyocytes. We present a case with evidence of apoptosis induced by myocardial ischemia without reperfusion. DNA fragmentation was demonstrated in the nuclei of the myocardial cells surrounding the ventricular septal rupture following acute myocardial infarction without reperfusion in the infarct-related left anterior descending coronary artery. This finding suggests that ischemia without reperfusion may induce apoptosis in myocardial cells. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) undergoes continuous remodeling during mammalian development. Although involvement of matrix metalloproteinases (MMPs) in ECM degradation has been well documented, how this process is regulated to allow proper ECM accumulation remains unclear. We previously showed the involvement of a membrane-anchored MMP regulator, RECK (reversion-inducing cysteine-rich protein with Kazal motifs), in vascular development in mice. Here we report that Reck mRNA can be detected in developing cartilage in E13.5∼16.5 mouse embryos and is progressively upregulated during differentiation of a chondrogenic cell line ATDC5 in vitro. In the early phase of ATDC5 differentiation, RECK expression stays low, multiple MMPs are upregulated, and there is ECM degradation at the sites of cellular condensation. In the later phase, RECK is upregulated inside the expanding cartilaginous nodules where type II collagen is accumulated while active ECM degradation persists along the rim of the nodules. Constitutive RECK expression suppressed initial cellular condensation, whereas RECK knockdown suppressed the later ECM accumulation in the cartilaginous nodules. These results suggest that RECK expression at the right place (in the core of the nodules) and at the right time (only in the later phase) is important for proper chondrogenesis and that RECK, together with MMPs, plays a crucial role in regulating dynamic processes of tissue morphogenesis. [ABSTRACT FROM AUTHOR]