The FGFR1 inhibitor PD173074 induces mesenchymal-epithelial transition through the transcription factor AP-1.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a crucial process in cancer progression that provides cancer cells with the ability to escape from the primary focus, invade stromal tissues and migrate to distant regions. Cell lines that lack E-cadherin show increased tumorigenesis and metastasis, and the expression levels of E-cadherin and Snail correlate inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (OSCC). Moreover, recent studies have shown that most EMT cases are regulated by soluble growth factors or cytokines. Among these factors, fibroblast growth factors (FGFs) execute diverse functions by binding to and activating members of the FGF receptor (FGFR) family, including FGFR1-4. Fibroblast growth factor receptor 1 is an oncoprotein that is involved in tumorigenesis, and PD173074 is known to be a selective inhibitor of FGFR1. However, the roles of FGFR1 and FGFR1 inhibitors have not yet been examined in detail. METHODS: Here, we investigated the expression of FGFR1 in head and neck squamous cell carcinoma (HNSCC) and the role of the FGFR1 inhibitor PD173074 in carcinogenesis and the EMT process. RESULTS: Fibroblast growth factor receptor 1 was highly expressed in 54% of HNSCC cases and was significantly correlated with malignant behaviours. Nuclear FGFR1 expression was also observed and correlated well with histological differentiation, the pattern of invasion and abundant nuclear polymorphism. Fibroblast growth factor receptor 1 was also overexpressed in EMT cell lines compared with non-EMT cell lines. Furthermore, treatment of HOC313 cells with PD173074 suppressed cellular proliferation and invasion and reduced ERK1/2 and p38 activation. These cells also demonstrated morphological changes, transforming from spindle- to cobble stone-like in shape. In addition, the expression levels of certain matrix metalloproteinases (MMPs), whose genes contain activator protein-1 (AP-1) promoter sites, as well as Snail1 and Snail2 were reduced following PD173074 treatment. CONCLUSION: Taken together, these data suggest that PD173074 inhibits the MAPK pathway, which regulates the activity of AP-1 and induces MET. Furthermore, this induction of MET likely suppresses cancer cell growth and invasion.

Associated demographics of persistent exhaled nitric oxide elevation in treated asthmatics.

BACKGROUND: The fraction of exhaled nitric oxide (FENO) is reduced by anti-inflammatory treatment in asthma. However, the FENO level is also regulated by individual demographics and there is considerable variation among clinically stable patients. OBJECTIVE: We hypothesized that some demographics may be responsible for persistent FENO elevation despite inhaled corticosteroids (ICS) therapy in asthma. METHODS: This was a prospective observational study. We initially screened 250 stable asthmatics and determined the FENO cut-off point for identifying poorly controlled asthma defined by one of the following criteria: Asthma control test <20, or forced expiratory volume in one-second % of predicted <80%, or peak expiratory flow variability <80% (Study 1). After 12-weeks, 229 patients who maintained high or low FENO were selected and the independent factors which might contribute to a high FENO were examined (Study 2). RESULTS: A FENO level >39.5 p.p.b. yielded 67% sensitivity and 76% specificity for identifying the patients with poorly controlled asthma. The persistent high FENO group (≥ 40 p.p.b.) was more likely to be ex-smokers, to show evidence of atopy (positive specific IgE, higher serum IgE and blood eosinophils), and to have allergic comorbidities. Especially, past smoking history, blood eosinophils, and chronic rhinosinusitis were identified to be independent predictors of high FENO. Neither the dose of ICS nor other medication use showed any difference between the groups. CONCLUSIONS AND CLINICAL RELEVANCE: These results suggested that past smoking history, blood eosinophilia, and chronic rhinosinusitis are involved in the persistent airway inflammation detected by FENO. Although their relative contributions on FENO values should be further quantified, clarification of the features of the subjects with high FENO might provide clues for adjustment of the treatment approach in asthma.

The effects of cigarette exposure on rat salivary proteins and salivary glands.

OBJECTIVE: Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on saliva and salivary glands (SGs). METHODS: Cigarette smoke-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (0 day), and 15 and 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. SGs were collected on 31 days. RESULTS: The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rates did not differ at 0, 15 or 30 days after the start of CS exposure. However, the amylase and peroxidase activities and total protein content in the saliva were significantly lower in 15-day CS-exposed rats than in 15-day control rats. Histological examination of the SGs of CS-exposed rats showed vacuolar degeneration, vasodilation and hyperemia. CONCLUSION: These results suggest that CS exposure has adverse impacts on salivary composition and SGs, which could aggravate the oral environment.

Professional healthcare use for life-threatening obstetric conditions.

Obstetric complications are the major causes of death and disability in women of reproductive age. Our study aimed at investigating the use of professional healthcare when women in communities recognised possible life-threatening obstetric conditions (PLTCs). We conducted a survey in a Cambodian district with a population of 130,000. The subjects were women of reproductive age who had delivered babies during a 3-month period prior to the survey. We interviewed 980 women, and 141 (14.4%) of these had PLTCs. The utilisation rates of professional healthcare were 47% for prolonged labour; 42% for bleeding during the delivery and puerperal period; 33% for antenatal bleeding; 25% for convulsion; and 23% for postpartum high fever. A logistic regression analysis revealed that education, geographic accessibility and parity were significant determinants of seeking healthcare. Two additional determinants, namely, economic affluence and antenatal care attendance, were identified in the socioeconomic status (SES) and obstetric models.

Intratumoural expression of thymidylate synthase is an independent predictor of prognosis in patients with squamous cell carcinoma of the tongue: results from a retrospective study.

The aim of this study was to assess the importance of immunohistochemical thymidylate synthase (TS) expression level as a prognostic marker in tongue cancer patients. In 140 patients with primary squamous cell carcinoma (SCC) of the tongue, intratumoural TS expression was evaluated by immunohistochemistry. The level of TS expression was determined by a semiquantitative scoring system, ranging from 1+ to 3+ according to the ratio of TS-positive cells. Of 140 patients, 64 (45.7%), 49 (35.0%) and 27 (19.3%) were assessed as 1+, 2+ and 3+, respectively. Univariate analyses demonstrated that both disease-free survival (DFS) and overall survival (OS) were significantly lower in patients with a TS 3+ tumour than in those with a TS 1+/2+ tumour (DFS: P = 0.0082, OS: P = 0.0100). In a multivariate analysis using the Cox regression model, cervical lymph-node status and TS expression level were selected as independent factors for DFS and OS. Maintenance adjuvant chemotherapy by oral 5-fluorouracil (5-FU) significantly improved DFS and OS in patients with a TS 1+/2+ tumour (DFS: P = 0.0027, OS: P = 0.0398). These data suggest that the level of immunohistochemical TS expression is an independent prognosticator in patients with tongue SCC, and may be useful in the selection of patients who would benefit from oral 5-FU adjuvant chemotherapy.

Inactivation of N-terminal signaling domain of Sonic hedgehog by forming a disulfide bond.

The N-terminal domain of mouse Sonic hedgehog (Shh-N) expressed in mammalian cells showed four-fold bands on non-reduced SDS-PAGE, though it was homogeneous under reduced conditions. It contains three cysteine residues, Cys-25, Cys-103, and Cys-184, which may be concerned with this heterogeneity. Therefore, we examined the formation of a disulfide bond in the recombinant Shh-N and identified three kinds of disulfides with a combination of peptide mapping and NH(2)-terminal amino acid sequencing analysis. Among them, one type of the Shh-N containing a disulfide bond of Cys-103/Cys-184 could be separated from the other Shh-Ns using reverse phase HPLC and had no activity of alkaline phosphatase induction in C3H10T1/2 cells. This molecule could also be made by denaturation of the purified Shh-N with guanidine-HCl under non-reduced conditions. On the other hand, the reduced Shh-N and the reduced S-methylated Shh-N at cysteine residues showed approximately 10-fold higher activity compared to the originally purified Shh-N. These results suggested that Shh-N was synthesized as an active form whose three cysteine residues did not form disulfide and inactivated finally by forming a disulfide bond between Cys-103 and Cys-184.

Prostaglandins affect the central nervous system to produce hyperglycemia in rats.

The influence of prostaglandins (PG) on central nervous system regulation of blood sugar homeostasis was studied in rats. Substances were injected into the third cerebral ventricle of anesthetized rats while rectal temperature and hepatic venous plasma glucose concentration were recorded. Stereotaxic microinjection of PGD2, E1, E2, and F2 alpha produced hyperglycemia and hyperthermia. The relative order of potency in hyperglycemia, PGF2 alpha greater than D2 greater than E1 greater than E2, was not consistent with that of hyperthermia, PGE2 greater than F2 alpha greater than E1 greater than D2, which suggests that hyperglycemia was a primary, not secondary, response to hyperthermia. Injection of PGF2 alpha caused a dose dependent (5-200 micrograms) increase in the hepatic venous plasma glucose level. Neither the injection of PGF2 alpha (50 micrograms) into the cortex nor into the systemic vein caused hyperglycemia. The injection of PGF2 alpha into the ventricle resulted in the increase of not only glucose, but also glucagon, epinephrine, and norephinephrine in the hepatic venous plasma. However, constant infusion of somatostatin through the femoral vein completely prevented the increase of glucagon after administration of PGF2 alpha, although the increase of plasma glucose level was still observed. PGF2 alpha-induced hyperglycemia did not occur in adrenodemedullated rats. Intravenous injection of naloxone or propranolol did not affect the hyperglycemia, but phentolamine significantly prevented the hyperglycemic effect of PGF2 alpha. These results suggest that intraventricular PGF2 alpha affects the central nervous system to produce hyperglycemia by increasing epinephrine secretion from the adrenal medulla.

Further analysis of cDNA clones for maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis. Nucleotide sequence of entire open reading frame and evidence for polyadenylation of mRNA at multiple sites in vivo.

Four clones of cDNA for phosphoenolpyruvate carboxylase [EC 4.1.1.31] were obtained from a maize green leaf cDNA library by colony hybridization. The largest cDNA was of full-length (3335 nucleotides), being 243 nucleotides longer than the cDNA cloned previously [(1986) Nucleic Acids Res. 14, 1615-1628]. Alignment of the sequence for the N-terminal coding region found in two of the four clones with the sequence reported previously, established the sequence of the entire coding region for the enzyme. The sequencing of 3'-untranslated region of the clones revealed that the poly(A) tract is attached at multiple sites in vivo.

The NH2-terminal region of the active domain of sonic hedgehog is necessary for its signal transduction.

The NH2-terminal domain of sonic hedgehog (residue 25-198) was expressed in both yeast and animal cells. The yeast-derived NH2-terminal domain of sonic hedgehog was less active by far than the animal cell-derived counterpart. The yeast-derived NH2-terminal domain of sonic hedgehog lacked 10 amino acids from the NH2-terminus. This cleavage of the yeast-derived NH2-terminal domain of sonic hedgehog might due to Kex 2. In contrast, a mutant yeast-derived NH2-terminal domain of sonic hedgehog (Lys-33 to Thr) retained its NH2-terminus and its activity was comparable to that of the animal cell-derived NH2-terminal domain of sonic hedgehog. The NH2-terminal deleted NH2-terminal domain of sonic hedgehog completely lost its activity, nevertheless it inhibited the alkaline phosphatase activity induced by the animal cell-derived NH2-terminal domain of sonic hedgehog in a dose-dependent manner. These data suggest that the NH2-terminal deleted NH2-terminal domain of sonic hedgehog retains a receptor-binding ability and that the NH2-terminal peptide of the NH2-terminal domain of sonic hedgehog is necessary for its signal transduction.

Maize phosphoenolpyruvate carboxylase involved in C4 photosynthesis: nucleotide sequence analysis of the 5' flanking region of the gene.

To clone the genomic DNA fragment containing the putative promoter region of the gene for phosphoenolpyruvate carboxylase [EC 4.1.1.31] involved in C4 photosynthesis (C4-type PEPC), maize genomic libraries were screened. On probing with a 384-bp fragment from the N-terminal coding region of the maize cDNA for C4-type PEPC, four EcoRI-fragments differing in the restriction map were cloned, reflecting the presence of a small gene family. Southern blot analyses were carried out on the genomic DNA and the cloned DNA fragments using several segments of the cDNA for C4-type PEPC as probes. The results indicated that the C4-type PEPC is encoded by a single gene and the cloned 7.0-kb fragment was derived from this gene. The transcription start site, as determined in a primer extension experiment, was located at about 4 kb downstream of the 5' end of the cloned fragment. The nucleotide sequence was determined for the region which extended about 1 kb upstream from the transcription start site and possible signal sequences related to gene expression were found, including four classes of direct repeats. The sequences of the corresponding regions of the other two cloned fragments (8.9 and 12.9 kb) which strongly hybridized with the 384-bp probe were very similar to each other, but they were quite different in the 5' upstream region from the sequence of the gene for C4-type PEPC.

Identification and characterization of positive regulatory elements in the human glyceraldehyde 3-phosphate dehydrogenase gene promoter.

The gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is expressed at high levels in almost all tissues. However, the molecular mechanism which sustains high-level expression of this house-keeping enzyme is still unknown. Here we show that transcriptional activity is reduced by deletion of the nucleotides from -181 to -144 (relative to the transcriptional start site) in the promoter of human GAPDH gene, both in CHO (derived from Chinese hamster ovary) and HepG2 (derived from human hepatoma) cells. Gel retardation assays revealed that at least two nuclear factors, termed GAPBF1 and GAPBF2, bind to this region. Mutations in the GAPBF1 binding site (-178 to -169) or the GAPBF2 binding site (-168 to -163) reduced this promoter activity in vivo, showing that these two sites contribute to the activity of the GAPDH gene promoter. Since mutations in the region from -162 to -146 also reduced the promoter activity, this region seemed to function as an added cis-element, although we failed to find a factor that interacted specifically with this region in vitro. Accordingly, we propose that there are multiple cis-elements in the region from -181 to -144, each of which contributes to the promoter activity of GAPDH gene; the GAPBF1 binding site has the unique feature of having a stretch of repeated A nucleotides.

Phosphoenolpyruvate carboxylase prevalent in maize roots: isolation of a cDNA clone and its use for analyses of the gene and gene expression.

In search of the gene family for phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31] in C4 plants, we isolated from a maize root cDNA library a new cDNA clone that cross-hybridized with a cDNA for PEPC involved in the C4 photosynthesis (Yanagisawa et al. (1988) FEBS Lett. 229, 107-110). Alignment of the nucleotide sequence with that of the probe cDNA revealed the absence of sequence homology in the 3' non-coding region between the two cDNAs. Southern blot hybridization probed with this specific sequence indicated that the corresponding gene is unique in the maize genome. Northern blot hybridization using the same probe showed that this gene-family member was expressed most strongly in roots and also in green leaves to a lesser extent, but not significantly in etiolated leaves.

Molecular evolution of phosphoenolpyruvate carboxylase for C4 photosynthesis in maize: comparison of its cDNA sequence with a newly isolated cDNA encoding an isozyme involved in the anaplerotic function.

Molecular events associated with the evolution of an enzyme for C4 photosynthesis were investigated. In maize, at least three isozymes of phosphoenolpyruvate carboxylase [EC 4.1.1.31] are known: the C4-form, the C2-form and the root-form, being named according to their physiological roles and pattern of tissue distribution [Ting, I.P. & Osmond, C.B. (1973) Plant Physiol. 51, 448-453]. A cDNA clone which presumably encodes the C3-form isozyme was newly isolated and analyzed. Comparison of the sequences of the C3-form and C4-form isozymes revealed that (i) the homologies in the nucleotide and deduced amino acid sequences were 71 and 77%, respectively, and (ii) the gene for the C4-form isozyme evolved under strong G/C pressure. The genes for these isozymes were found to be located apart on different chromosomes. A phylogenetic tree was constructed using 8 amino acid sequences of phosphoenolpyruvate carboxylases from various sources. The topology of the tree indicated that, at least in monocots such as maize and sorghum, the genes for the C4-form and C3-form isozymes diverged from their common ancestral gene earlier than the monocot-dicot divergence (about 2 x 10(8) yr ago), though the divergence of maize (C4 plant) from wheat (C3 plant) is supposed to have occurred much later (6 x 10(7) yr ago).

Asymmetric synthesis of the core structure of (-)-CP-263,114.

The enantioselective construction of a fully functionalized core structure of (-)-CP-263,114 (1), containing most of the required functionality for total synthesis, was conducted through sequential radical fragmentation-reductive olefination.

Expression of cyclin D1 and GSK-3beta and their predictive value of prognosis in squamous cell carcinomas of the tongue.

The status of cyclin D1 and glycogen synthase kinase 3beta (GSK-3beta) was investigated in 41 patients with T1 and T2 squamous cell carcinomas (SCCs) of the tongue. Out of the 41 SCCs, 27 (65.9%) showed overexpression of cyclin D1 in comparison with normal lingual epithelia by an immunohistochemical method. Cyclin D1 gene amplification was detected in only two (9.1%) of 22 informative cases of the SCCs by differential PCR. Expression of GSK-3beta, which was found to regulate proteosomal degradation of cyclin D1 protein, was reduced in 16 cases (39.0%) of the SCCs relative to normal epithelia, and the intensity of GSK-3beta staining showed an inverse association with cyclin D1. These findings suggest that overexpression of cyclin D1 primarily results from stabilization due to reduction of GSK-3beta, but not cyclin D1 gene amplification, in lingual SCCs. Kaplan-Meier analysis demonstrated that the patients with high cyclin D1 and reduced GSK-3beta expression had a significantly lower 5-year survival than the patients with low cyclin D1 and non-reduced GSK-3beta expression (P=0.014). The cyclin D1 and GSK-3beta coupled assessment was more valuable for the prediction of prognosis than assessment based on cyclin D1.

Schwannoma of the lesser omentum.

A case of solitary benign schwannoma of the omentum is detected by CT, ultrasonography and angiography, as a solid mass containing cystic regions.

Biosynthetic intermediates of the tetradehydro cyclic dipeptide albonoursin produced by Streptomyces albulus KO-23.

The cell-free extract of an albonoursin-producing strain Streptomyces albulus KO-23 catalyzes the conversion of cyclo(L-Leu-L-Phe) (1) to albonoursin (2). At the early stage of this conversion, two compounds were newly formed prior to albonoursin synthesis in the reaction mixture. These compounds were isolated and identified as (Z)-3-benzylidene-6-isobutyl-2,5-piperazinedione (4) and (Z)-3-benzyl-6-isobutylidene-2,5-piperazinedione (3). The cell-free extract also catalyzed the conversion of compound 3 or 4 to albonoursin. From these results, albonoursin was found to be biosynthesized via these compounds from cyclo(L-Leu-L-Phe). These didehydro diketopiperazines exhibited no inhibitory activity toward the first cleavage of sea urchin embryo in contrast to the higher cytotoxicity for albonoursin, indicating that dehydrogenation at alpha,beta-positions of both amino acid residues in diketopiperazines is required for cytotoxicity.

Presurgical evaluation for dental implants using a reformatting program of computed tomography: maxilla/mandible shape pattern analysis (MSPA).

A new analysis technique that provides a shape pattern of the maxilla or mandible is described. This technique uses a software program of computed tomography multiplanar reformation (CT/MPR). The successive cross-sectional images obtained by the CT/MPR are numbered, and the height and width of the maxilla and mandible at each of these cross-sectional images at specified levels are measured. The measured height and width are plotted against the number of the cross-section. This technique, termed maxilla/mandible shape pattern analysis (MSPA), creates an easy profile of the shape of the maxilla and mandible, and it enhances treatment planning for the placement of dental implants.