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.

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.

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.

Synthesis of 2-deoxy-2-[(2,2-difluoro-3-hydroxytetradecanoyl)amino]-3-O- [(R)-3-(tetradecanoyloxy)tetradecanoyl]-D-glucopyranose 4-phosphate.

2-Deoxy-2-[(2,2-difluoro-3-hydroxytetradecanoyl)amino]-3-O-[(R)-3- (tetradecanoyloxy)tetradecanoyl]-D-glucopyranose 4-phosphates (9H,L) were synthesized from allyl 2-amino-2-deoxy-4,6-O- isopropylidene-beta-D-glucopyranoside (1), (+/-)-3-[(benzyloxycarbonyl)oxy]-2,2-difluorotetradecanoic acid, and (R)-3- (tetradecanoyloxy)tetradecanoic acid. Both compounds 9H and 9L were more active than GLA-60 for the prostaglandin D2 releasing test on macrophages.

Proliferation and differentiation of erythroleukemia cell line (ELM-I-1) in response to erythropoietin and interleukin 3.

Here we describe the characterization of Epo-responsive mouse erythroleukemia cell line ELM-I-1. ELM-I-1 cells possess Epo binding sites on their membranes and differentiate into hemoglobin-positive cells when cultured in the presence of Epo. About 20% of the cells were hemoglobin-positive after a 3- to 4-day exposure to recombinant human Epo in liquid culture. Supplementation of recombinant mouse IL-3 during culture had an augmentative effect on Epo-mediated differentiation, although IL-3 alone did not induce differentiation. Both Epo and IL-3 stimulated the growth of ELM-I-1 cells, and their effects were a slightly additive. These findings indicate that ELM-I-1 cells are suitable for studying the interaction between Epo and IL-3 in erythroid differentiation at a subcellular level. ELM-I-1 may also offer a valuable bioassay system for Epo.

Formation of lambda transducing phage in vitro: involvement of DNA gyrase.

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.

The complete amino acid sequence of rice bran trypsin inhibitor.

The complete amino acid sequence of a double-headed trypsin inhibitor (RBTI) from rice bran was determined by a combination of limited proteolysis of the native inhibitor with Streptomyces griseus trypsin at pH 3 and conventional methods. RBTI consists of 133 amino acid residues including 18 half-cystine residues which are involved in 9 disulfide bridges in the molecule. The limited proteolysis at pH 3 produced a major split of Lys(83)-Met(84) and a minor split of Arg(107)-Val(108) together with a non-enzymatic hydrolysis of Asp(19)-Pro(20) in the molecule. The established sequence showed that RBTI is composed of 4 domains, domains I and III, and domains II and IV being homologous to the first and the second domains of soybean Bowman-Birk inhibitor, respectively, indicating that RBTI has a duplicated structure of the Bowman-Birk type inhibitor.

Intercellular junctions in embryonic chick cardiac muscle revealed by rapid freezing and freeze-substitution.

Using the method of rapid freezing and freeze-substitution, the embryonic chick cardiac muscle was investigated by transmission electron microscopy. Initially, the intercellular junctional complexes (fasciae adherentes and desmosomes) were formed in close proximity to each other along a nearly straight line. Subsequently, the separation of fasciae from desmosomes took place to form intercalated discs. The cell membranes of fasciae adherentes were reinforced with highly interwoven fine fibrils at which myofibrils terminated. The intercellular space of fasciae was bridged with fine fibrillar structures seemingly connected by a thin line at their middle portions. In the intercellular space of desmosomes, central lamina and traversing filaments were clearly observed. The outer and inner leaflets of the desmosomal plasmalemma were asymmetrically differentiated; the outer leaflet was thinner than the inner leaflet. On the inner side of the cell membrane, an electron-lucent layer and a dense desmosomal plaque were observed. The latter structure had protrusions with less electron density towards the cytoplasmic side. Further inside, a meshwork of fine fibrils was seen along and toward which bundles of intermediate filaments ran. The results obtained with freeze-substitution appeared to provide more information than those with thin sections after conventional fixation or with replicas of chemically fixed/glycerinated or physically fixed/deep-etched materials.

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 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.

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 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 a 3-ether analogue of lipid A.

Lipid A 3-ether analogues were synthesized from allyl 2-deoxy-4,6-O-isopropylidene-2-trifluoroacetamido-alpha-D-glucopyr anoside and 3,4,6-tri-O-acetyl-2-trifluoroacetamido-alpha-D-glucopyranosyl bromide. The compound lost completely the endotoxic activity.

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.

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 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.

Syntheses and biological activities of new carbapenem derivatives.

3-beta-Alanyloxymethyl, 3-glycyloxymethyl and 3-methyl derivatives of 6-(1-hydroxyethyl)-carbapenems, (VIIa, VIIb and VIII), and 3-methyl-6-(1-hydroxyethyl)carbapenam (IX) were synthesized from 3-(1-tert-butyldimethylsilyloxyethyl)-4-(3-chloro-2-oxopropyl) -2-azetidinone (I). The antibacterial activities of these compounds proved that the delta 2 double bond was essential for the appearance of bioactivity, whereas the amino group on the C-3 side chain was not necessary.