NOTCH1 expression predicts patient prognosis in esophageal squamous cell cancer.

PURPOSE: The prognosis of patients with esophageal cancer remains poor, and the classification of tumor node metastasis has proven insufficient to predict patient prognosis. Therefore, novel predictive markers of esophageal cancer prognosis are needed. Notch receptors and their ligands have been reported to be upregulated in cervical, lung, colon, renal, and pancreatic cancers, but NOTCH1 expression has not been studied in esophageal cancer. METHODS: Expression of NOTCH1 was quantified by real-time reverse transcription-polymerase chain reaction in 55 primary esophageal squamous cell carcinomas (ESCCs) and their paired normal esophageal mucosa. We then examined the correlations between NOTCH1 expression, clinicopathological factors, and prognosis in patients with ESCC. RESULTS: The probability of overall survival was significantly lower for patients with high NOTCH1 expression (p = 0.0028; log-rank test). Overexpression of NOTCH1 was identified as a significant and independent prognostic factor (p = 0.0061) in patients who had undergone surgical treatment for ESCCs. The hazard ratio for predicting early death was 4.298 (95% confidence interval 1.515-12.195) for high versus low NOTCH1 expression. CONCLUSIONS: Our data indicate that NOTCH1 may be a candidate molecular prognostic marker and a molecular target for the development of an effective therapeutic intervention for patients with ESCC.

Morphological and biochemical signs of age-related neurodegenerative changes in klotho mutant mice.

Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported.

Measuring the regret of bereaved family members regarding the decision to admit cancer patients to palliative care units.

OBJECTIVE: The purposes of this study were to develop a bereaved family regret scale measuring decision-related regret of family members about the admission of cancer patients to palliative care units (PCUs) and to examine the validity and reliability of this scale. METHOD: Bereaved families of cancer patients who had died in one regional cancer center from September 2004 to February 2006 received a cross-sectional questionnaire by mail. The questionnaire contained seven items pertaining to decision-related regret about the patient's admission to the PCU, the Care Evaluation Scale (CES), an overall care satisfaction scale, and a health-related quality-of-life (QOL) scale (SF-8). One month after receiving a completed questionnaire, we conducted a retest with the respondent. RESULTS: Of the 216 questionnaires successfully mailed to the bereaved families, we received 137 questionnaires and were able to analyze the responses for 127 of them, as the other 10 had missing data. By exploratory factor analysis and confirmatory factor analysis, we identified two key factors: intrusive thoughts of regret and decisional regret. This scale had sufficient convergent validity with CES, overall care satisfaction, SF-8, sufficient internal consistency, and acceptable test-retest reliability. CONCLUSION: We have developed and validated a new regret scale for bereaved family members, which can measure their intensity of regret and their self-evaluation about their decision to admit their loved ones to PCUs.

Synthesis of hydantocidin and C-2-thioxo-hydantocidin.

Hydantocidin, a naturally occurring strong herbicide, was synthesized in an overall yield of 35.2%, with the accompanying 1'-epi-hydantocidin in overall 9.6% yield from 2,3-O-isopropylidene-D-ribono-1,4-lactone. C-2-thioxo-hydantocidin and its spiro-epimer were also synthesized in an overall yield of 14.4% and 8.5%, respectively.

Synthesis and biological activity of 4',8-dihydroxyisoflavon-7-yl D-hexopyranosides.

Four 4',8-dihydroxyisoflavon-7-yl hexopyranoside derivatives having an aglycon part of A-76202 were synthesized, and their biological activities were evaluated toward rat liver alpha-glucosidase. However, the activities were disappointing.

Synthesis of lipid A type pyran carboxylic acids with ether chains and their biological activities.

Synthesis of lipid A type pyran carboxylic acids having ether chains at both the C-3' and C-4 positions and their bioactivities toward human U937 cells are described.

Synthesis of GLA-60 type pyran carboxylic acids with an alkyl chain instead of an ester chain as LPS-antagonists.

Synthesis of GLA-60 type pyran carboxylic acid analogues with an alkyl chain instead of an ester chain and their LPS-antagonist activity toward human U937 cells are described.

Synthesis and biological activities of lipid A-type pyrancarboxylic acid derivatives.

The synthesis of lipid A-type pyrancarboxylic acid derivatives, which have a carboxylic acid group in the anomeric position of the reducing part of the disaccharide instead of the phosphate group in lipid A, is described. One of the compounds thus synthesized, which has an acyl substitution pattern similar to that of Escherichia coli lipid A, showed lipopolysaccharide (LPS)-agonistic activity. The other, which contains four lipid chains in the molecule, exhibited strong LPS-antagonistic activity toward human monoblastic U937 cells.

Overexpression of rck/p54, a DEAD box protein, in human colorectal tumours.

The RCK gene is a target of the t(11;14)(q23;q32) chromosomal translocation observed in human B-cell lymphoma, and the overexpression of its protein (rck/p54) by the translocation was shown to cause malignant transformation. The rck/p54 protein belongs to the DEAD box protein/RNA helicase family, which has a variety of functions such as translation initiation, pre-mRNA splicing and ribosome assembly. The expression of rck p54 in colorectal adenocarcinoma cells was examined by immunohistochemistry and Western blot analysis. The rck/p54 protein was found to be overexpressed in tumour tissues resected from 13 (50%) out of 26 cases of colorectal adenocarcinomas and two out of two (100%) cases of colonic severe dysplastic adenomas. In view of activities of rck/p54 determined in other tissue types, we suggest that rck/p54 may contribute to the cell proliferation and carcinogenesis at the translational level in the development of colorectal tumours.

Heat stress activates fission yeast Spc1/StyI MAPK by a MEKK-independent mechanism.

Fission yeast Spc1/StyI MAPK is activated by many environmental insults including high osmolarity, oxidative stress, and heat shock. Spc1/StyI is activated by Wis1, a MAPK kinase (MEK), which is itself activated by Wik1/Wak1/Wis4, a MEK kinase (MEKK). Spc1/StyI is inactivated by the tyrosine phosphatases Pyp1 and Pyp2. Inhibition of Pyp1 was recently reported to play a crucial role in the oxidative stress and heat shock responses. These conclusions were based on three findings: 1) osmotic, oxidative, and heat stresses activate Spc1/StyI in wis4 cells; 2) oxidative stress and heat shock activate Spc1/StyI in cells that express Wis1AA, in which MEKK consensus phosphorylation sites were replaced with alanine; and 3) Spc1/StyI is maximally activated in Deltapyp1 cells. Contrary to these findings, we report: 1) Spc1/StyI activation by osmotic stress is greatly reduced in wis4 cells; 2) wis1-AA and Deltawis1 cells have identical phenotypes; and 3) all forms of stress activate Spc1/StyI in Deltapyp1 cells. We also report that heat shock, but not osmotic or oxidative stress, activate Spc1 in wis1-DD cells, which express Wis1 protein that has the MEKK consensus phosphorylation sites replaced with aspartic acid. Thus osmotic and oxidative stress activate Spc1/StyI by a MEKK-dependent process, whereas heat shock activates Spc1/StyI by a novel mechanism that does not require MEKK activation or Pyp1 inhibition.

Activin A: a commitment factor in erythroid differentiation.

Erythropoietin is known to be an essential hemopoietic growth factor for maturation of erythroid progenitor cells. Like other hemopoietic growth factors, erythropoietin acts as a survival factor that supports maturation of the erythroid progenitor through the suppression of apoptosis. It is unclear whether erythropoietin can also induce differentiation, or if another external regulator is needed to initiate this process. The present study using murine cell lines revealed that maturation of the erythroid lineage requires costimulation by activin A and erythropoietin. Erythropoietin alone dose not induce differentiation and cells stimulated by activin A alone undergo apoptotic death. Costimulation with erythropoietin and activin A, however, rescues the cells from apoptotic death and permits differentiation. Two-step cultivation showed that cells pretreated with activin A no longer need activin A and differentiate in the presence of erythropoietin alone. The action of activin A commits the cell to death or to differentiation, and the presence of erythropoietin enables differentiation through suppression of apoptosis.

Mcs4 mitotic catastrophe suppressor regulates the fission yeast cell cycle through the Wik1-Wis1-Spc1 kinase cascade.

Spc1 in Schizosaccharomyces pombe is a member of the stress-activated protein kinase family, an evolutionary conserved subfamily of mitogen-activated protein kinases (MAPKs). Spc1 is activated by a MAPK kinase homologue, Wis1, and negatively regulated by Pyp1 and Pyp2 tyrosine phosphatases. Mutations in the spc1+ and wis1+ genes cause a G2 cell cycle delay that is exacerbated during stress. Herein, we describe two upstream regulators of the Wis1-Spc1 cascade. wik1+ (Wis1 kinase) was identified from its homology to budding yeast SSK2, which encodes a MAPKK kinase that regulates the HOG1 osmosensing pathway. Delta wik1 cells are impaired in stress-induced activation of Spc1 and show a G2 cell cycle delay and osmosensitive growth. Moreover, overproduction of a constitutively active form of Wik1 induces hyperactivation of Spc1 in wis1(+)-dependent manner, suggesting that Wik1 regulates Spc1 through activation of Wis1. A mutation of mcs4+ (mitotic catastrophe suppressor) was originally isolated as a suppressor of the mitotic catastrophe phenotype of a cdc2-3w wee1-50 double mutant. We have found that mcs4- cells are defective at activation of Spc1 in response to various forms of stress. Epistasis analysis has placed Mcs4-upstream of Wik1 in the Spc1 activation cascade. These results indicate that Mcs4 is part of a sensor system for multiple environmental signals that modulates the timing of entry into mitosis by regulating the Wik1-Wis1-Spc1 kinase cascade. Inactivation of the sensor system delays the onset of mitosis and rescues lethal premature mitosis in cdc2-3w wee1-50 cells.

Nitric oxide donor SNAP induces apoptosis in smooth muscle cells through cGMP-independent mechanism.

Recent evidence suggests that nitric oxide (NO) may function as a second messenger in the intracellular signal transduction pathways. We explored the possibility that NO was involved in the signal for triggering apoptosis in smooth muscle cells (SMCs). Chemical NO donors induced SMCs apoptosis in a concentration- and time-dependent manner. The membrane-permeable cGMP analogue, dibutyryl-cGMP, did not induce SMCs apoptosis, and the highly selective inhibitor of cGMP-dependent protein kinase, KT5823, was unable to inhibit the induction of NO-induced SMCs apoptosis. Inhibitor of ADP-ribosyltransferase slightly attenuated the induction of SMCs apoptosis by S-nitroso-N-acetyl penicillamine (SNAP). The inhibitor of Na+-H+ antiporter, amiloride, completely inhibited the induction of SMCs apoptosis by SNAP. These results demonstrate for the first time that NO can induce apoptosis in SMCs, suggesting that NO acts as a mediator in the development of atherosclerosis lesion via alterations in the number of SMCs. In addition, the results suggest that NO exert these effects through a pathway that does not involve guanylate cyclase and cGMP-dependent protein kinase.

Syntheses of 1-O-carboxyalkyl GLA-60 analogues.

As part of our ongoing study to survey potent LPS antagonists, the following six compounds were synthesized in an efficient manner: 3-carboxypropyl and carboxymethyl 2-deoxy-2-(2,2-difluorotetradecanamido)-4-O-phosphono-3-O-[(R)-3- (tetradecanoyloxy)tetradecanoyl]-alpha- and beta-D-glucopyranosides (11 and 23; 32 and 36), as well as the non-fluorinated equivalents, carboxymethyl 2-deoxy-4-O-phosphono-2-tetradecanamido-3-O-[(R)-3-(tetradecano yloxy)- tetradecanoyl]-alpha-D-glucopyranoside (44) and carboxymethyl 2-deoxy-2-[(R)-3-(hydroxy)tetradecanamido]-4-O-phosphono-3-O-[(R)- 3- (tetradecanoyloxy)tetradecanoyl]-alpha-D-glucopyranoside (48). Of these compounds, 32 was most pronounced in terms of LPS-antagonistic activity.

Anti-activin A antibody (IgY) specifically neutralizes various activin A activities.

Activin A (beta A beta A), originally isolated from ovarian follicular fluids as a follicule-stimulating hormone (FSH) secretion stimulator, has also been identified as an erythroid differentiation factor (EDF), a neuron survival factor and a mesoderm-inducing factor. Thus, activin A is a multifunctional factor, and further studies on its physiological function are important. However, it is very difficult to produce a specific antibody to neutralize the activity of activin A because of its highly conserved amino acid sequence across mammalian species. In this study, we succeeded in generating an antibody against activin A, which can neutralize several activities of activin A, such as the stimulation of FSH secretion from pituitary cells and the induction of the differentiation of erythrocytes in vitro. This antibody did not affect the activity of activin B (beta B beta B), which induces the differentiation of erythrocytes in vitro, and the activity of inhibin A (alpha beta A), which inhibits FSH secretion from pituitary in vitro, but slightly neutralized that of activin AB (beta A beta B). Western blotting analysis showed that this antibody recognized both dimeric and monomeric forms of the beta A subunit of activin and inhibin. These results suggest that this antibody recognizes the beta A subunit of activin and specifically neutralizes the activity of a dimer of the beta A subunit, activin A. Furthermore, by the addition of this antibody to the culture medium, the development of murine embryos was suppressed, suggesting that endogenous activin A plays an important role in murine development. These results indicate the usefulness of this antibody for studies of endogenous activin actions.

Syntheses of 1-O-[5-(carboxy)pentanoyl]-2-deoxy-2-(2,2- difluorotetradecanamido)-3-O-[(R)-3-(tetradecanoyloxy)tetradecanoyl]-4- O- phosphono-alpha-D-glucopyranose and its analogues.

1-O-[5-(Carboxy)pentanoyl]-2-deoxy-2-(2,2-difluorotetradecanamido) -3-O- [(R)-3-(tetradecanoyl-oxy)tetradecanoyl]-4-O-phosphono-alpha-D-glucop yranos e (13) and its analogues (16 and 19) were synthesized. Compound 13 showed strong LPS-agonistic activity.

Synthesis of 5-epi-trehazolin (trehalostatin).

Synthesis of 5-epi-trehazolin (trehalostatin) (2) was accomplished via the crucial intermediate, epoxide (6 alpha), from D-glucose. The stereochemistry of epoxide (6 alpha) and its isomer (6 beta) which were obtained from Sharpless epoxidation, was determined by comparison between the NMR relaxation times of relevant protons.

Synthesis of trehazolin beta-anomer.

Synthesis of trehazolin beta-anomer (3) from a D-glucose derived azido alcohol (4), was accomplished. 2-Chloro-1-methylpyridinium iodide was used in place of 2-chloro-3-ethylbenzoxazolium tetrafluoroborate as a means of preventing concomitant anomerization. Evaluation of compound (3) reveals that the stereochemistry of the anomeric position is significant for generation of inhibitory activities towards trehalases.

Synthesis of 2-deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]- 3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and its (2S,3R)-isomer.

2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[( 3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and its (2S,3R)-isomer were respectively synthesized from allyl 2-[(2R,3S)-3-(benzyloxycarbonyloxy)-2-fluorotetradecanamido] -2-deoxy-4,6-O-isopropylidene-beta-D-glucopyranoside and its corresponding (2S,3R)-isomer. Both target compounds did not activate macrophage, but the (2S,3R)-analogue strongly inhibited the binding of LPS to macrophage.

Distribution of connectin (titin) and transverse tubules at myotendinous junctions.

The ends of muscle fibers form many longitudinal projections which are further divided into numerous processes and attach to the collagen fibrils of tendons to form myotendinous junctions (MTJs). Immunocytochemical and electron microscopic observations on pectoralis muscles of the chicken revealed the presence of an elastic filamentous protein, connectin (titin), within the terminal sarcomere on the side adjacent to the terminal Z bands, and the absence of connectin and myosin and the presence of actin at the apical sarcoplasmic region of MTJ processes between the terminal Z band and the MTJ sarcolemma. Intermediate voltage electron microscopy showed that T tubules in the terminal sarcomere were absent at the level of the A-I junction on the MTJ side in the rat vastus intermedius, and at the level of the terminal Z band or under the MTJ subsarcolemmal densities in the chicken pectoralis.