Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Expression of the yes proto-oncogene in cerebellar Purkinje cells.

To identify the kinds of cells in the brain that express the yes proto-oncogene, we examined chicken brains by using immunofluorescent staining and in situ hybridization. Both approaches showed that the highest level of the yes gene product was in cerebellar Purkinje cells. In addition, we analyzed Purkinje cell degeneration (pcd) mutant mice. The level of yes mRNA in cerebella of pcd mutants was four times lower than that found in cerebella of normal littermates. Our studies point to Purkinje cells as an attractive model for functional studies of the yes protein.     

Structure-based design,synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC₅₀ = 1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC₅₀ = 5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C] = 4%, po, 5 mg/kg, once-daily) and COLO205 (T/C = 13%, po, 15 mg/kg, once-daily) mouse xenograft models.     

Structural requirement of leucine for activation of p70 S6 kinase

The addition of leucine induced activation of p70S6k in amino acid-depleted H4IIE cells. Whereas the activation of p70S6k by leucine was transient, the complete amino acid stimulated p70S6k more persistently. The effect of leucine on p70S6k was sensitive to rapamycin, but less sensitive to wortmannin. Using various amino acids and derivatives of leucine, we found that the chirality, the structure of the four branched hydrocarbons, and the primary amine are required for the ability of leucine to stimulate p70S6k, indicating that the structural requirement of leucine to induce p70S6k activation is very strict and precise. In addition, some leucine derivatives exhibited the ability to stimulate p70S6k and the other derivatives acted as inhibitors against the leucine-induced activation of p70S6k.     

Expression of cys-cys chemokine ligand 21 on human gingival lymphatic vessels

The cys-cys (C-C) chemokine ligand 21 is a member of the C-C chemokines that constitute a group of heparin-binding cytokines with a pattern of four or six conserved cysteines. The CCL21 is known to be expressed in secondary lymphoid tissues, however it has rarely been reported for the expression on peripheral lymphatic vessels in somatic tissue. Here we investigated the expression of CCL21 on lymphatic vessels identified by anti-desmoplakin in uninflamed and inflamed human gingiva. In uninflamed tissue the expression of CCL21 was detected on lymphatic vessels in gingiva. In uninflamed gingiva the expression of CCL21 was detected on all lymphatic capillaries of the mucosal connective tissue papillae. There were two types of collecting lymphatic vessels in the lamina propria mucosae expressing CCL21 strongly or very weakly. In inflamed gingiva no expression of CCL21 was detected on lymphatic vessels. In all tissue sections no blood vessels expressing CCL21 were observed. These results may suggest that the expression of CCL21 is predominantly induced in the peripheral lymphatic endothelium of the uninflamed mucosal microcirculation, and that under inflamed conditions a reduction of CCL21 occurs in lymphatic endothelium.     

LET dependence of lethality in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> irradiated by heavy ions

To clarify the effect of heavy ions in plants, dry seeds of Arabidopsis were irradiated with carbon, neon, and argon ions with various linear energy transfer (LET) values. The relative biological effectiveness (RBE) for lethality peaked at LET values over 350 keV/microns for neon and argon ions. This LET giving the peak RBE was higher than the LET of 100-200 keV/microns which was reported to have a maximum RBE for other types of cells, such as mammalian cells. Furthermore, sterility showed a higher RBE at an LET of 354 keV/microns with neon ions than that at an LET of 113 keV/microns with carbon ions. Lethality and sterility are both considered to be caused by damage to DNA. The results indicate that the LET having a maximum of RBE for lethality is higher in Arabidopsis seeds than in other unicellular systems. The most likely explanation for this shift of LET is that the DNA in dry seeds has a different chemical environment and/or hydration state than the DNA in cells in culture.     

Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

Production of human growth hormone in transgenic rice seeds: co-introduction of RNA interference cassette for suppressing the gene expression of endogenous storage proteins

Rice seeds are potentially useful hosts for the production of pharmaceutical proteins. However, low yields of recombinant proteins have been observed in many cases because recombinant proteins compete with endogenous storage proteins. Therefore, we attempt to suppress endogenous seed storage proteins by RNA interference (RNAi) to develop rice seeds as a more efficient protein expression system. In this study, human growth hormone (hGH) was expressed in transgenic rice seeds using an endosperm-specific promoter from a 10 kDa rice prolamin gene. In addition, an RNAi cassette for reduction of endogenous storage protein expressions was inserted into the hGH expression construct. Using this system, the expression levels of 13 kDa prolamin and glutelin were effectively suppressed and hGH polypeptides accumulated to 470 μg/g dry weight at the maximum level in transgenic rice seeds. These results suggest that the suppression of endogenous protein gene expression by RNAi could be of great utility for increasing transgene products.     

LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium.

We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kappaB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kappaB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC.