Release of fibroblast growth factor-1 by human squamous cell carcinoma correlates with autocrine cell growth

Summary A squamous cell carcinoma cell line Nakata proliferated in serum-free culture and was not responsive to exogenous fibroblast growth factor-1 (FGF-1). Immunostaining revealed that Nakata cells expressed FGF-1 in their cytoplasms and nuclei. Two molecular mass species of FGF-1 (16 and 18 kDa) were identified in cell extracts by Western blot. These cells also expressed high-affinity FGF-1 binding sites (Kd=360 pM, 28 000 sites/cell). The results of cross-linking with [¹²⁵I]FGF-1 demonstrated the presence of two bands with molecular masses of 160 and 140 kDa. The addition of FGF-1 specific antisense oligonucleotides at 25 μM to Nakata cells resulted in an 82% inhibition in cell growth and suppressed FGF-1 expression. This effect was dose-dependent and specific, because sense oligonucleotides were ineffective in inhibiting cell growth. In addition, Nakata cell growth was suppressed by an anti-FGF-1 neutralizing antibody, which resulted in a 52% inhibition at 8 μg/ml. These results demonstrate that Nakata cells produce FGF-1, and indicate that this growth factor acts in an autocrine manner by interacting with FGF-1 binding sites on Nakata cells.     

Effect of pH, carbamylation and other hemoglobins on deoxyhemoglobin S aggregation inside intact erythrocytes as detected by proton relaxation rate measurements.

Measurement of the transverse water proton relaxation rate has been used to study the effect of pH, carbamylation, and other hemoglobins on the aggregation of deoxyhemoglobin S inside intact erythrocytes. Upon complete deoxygenation, cyanate-treated ($\text{S}\text{S}$) erythrocytes and erythrocytes heterozygous with respect to hemoglobin S ($\text{A}\text{S}$, $\text{C}\text{S}$, and $\text{S}\text{D}$) have high transverse water proton relaxation rates very similar to the values obtained with homozygous ($\text{S}\text{S}$) erythrocytes. These results suggest extensive intermolecular interactions between deoxyhemoglobin S molecules and a resultant increase in the correlation time for the small fraction of “irrotationally bound” water. When the transverse relaxation rate in deoxygenated ($\text{S}\text{S}$) erythrocytes was measured as a function of pH, the maximum rate was observed between pH 7.0 and 7.5. Upon increasing the pH beyond this range the observed relaxation rate decreases as does the number of sickled cells. Upon decreasing the pH, the observed transverse relaxation rate also decreases but the ratio of values from $\text{deoxy}\text{oxy}$ ($\text{S}\text{S}$) erythrocytes remains in the normal range of 4–6 and the number of sickled cells does not change. Therefore, the deoxyhemoglobin S aggregate inside sickled erythrocytes, as observed by water proton relaxation rates, is not altered by carbamylation or by the presence of nongelling hemoglobins. In addition, the enhancement of the relaxation rates as a function of pH is consistent with the number of sickled forms observed.     

Cyanidioschyzon merolae ferredoxin: a high resolution crystal structure analysis and its thermal stability

Cyanidioschyzon merolae (Cm) is a single-cell red alga that grows under moderately thermophilic (40-50°C), acidic (pH 1-3) conditions. We purified a Cm ferredoxin (Fd) that was characterized as a plant-type [2Fe-2S] Fd by physicochemical techniques. X-ray crystallography revealed that the overall three-dimensional structure of CmFd was highly similar to, but slightly different from, the [2Fe-2S] Fd from Spinacia oleracea, whose growth temperature is 15-20°C. Therefore, slight structural differences, including non-covalent-bond number and amino acid sequence, may underlie the differential thermostabilities of the plant-type Fds.     

C17,20-lyase inhibitors I. Structure-based de novo design and SAR study of C17,20-lyase inhibitors

Novel nonsteroidal C(17,20)-lyase inhibitors were synthesized using de novo design based on its substrate, 17 alpha-hydroxypregnenolone, and several compounds exhibited potent C(17,20)-lyase inhibition. However, in vivo activities were found to be short-lasting, and in order to improve the duration of action, a series of benzothiophene derivatives were evaluated. As a result, compounds 9h, (S)-9i, and 9k with nanomolar enzyme inhibition (IC(50)=4-9 nM) and 9e (IC(50)=27 nM) were identified to have powerful in vivo efficacy with extended duration of action. The key structural determinants for the in vivo efficacy were demonstrated to be the 5-fluoro group on the benzothiophene ring and the 4-imidazolyl moiety. Superimposition of 9k and 17 alpha-hydroxypregnenolone demonstrated their structural similarity and enabled rationalization of the pharmacological results. In addition, selected compounds were also identified to be potent inhibitors of human enzyme with IC(50) values of 20-30 nM.     

Localization ofd-amino acid oxidase mRNA in the mouse kidney and the effect of testosterone treatment

d-Amino acid oxidase (DAO), which catalyzes oxidative deamination ofd-amino acids, is known to be highly expressed in the kidney. This study was designed to examine the localization of DAO mRNA in the mouse kidney using in situ hybridization histochemistry (ISH). For comparison, ISH for mRNA of ornithine decarboxylase (ODC), which is also highly expressed in the mouse kidney, was simultaneously performed. Adult, male mice which received 1 mg of testosterone propionate or vehicle injection, were sacrificed 14 h after injection and their kidneys were removed and processed for ISH. Hybridization signals for both mRNAs were exclusively located over the epithelial cells of the proximal tubule in the vehicle-treated animals. Signals for the DAO mRNA were observed at nearly the same hybridization intensity throughout the proximal tubule, whereas hybridization signals for the ODC mRNA were observed exclusively in the pars convoluta. Following testosterone treatment, ODC mRNA in the pars convoluta was expressed with a stronger intensity than that in the vehicle-injected animals. ODC mRNA was also expressed in the pars recta with a weaker intensity than in the pars convoluta. On the other hand, DAO mRNA expression was little affected by testosterone treatment. These results indicate that, although both genes are possibly expressed in the same cells, the expression of these genes is regulated by different mechanisms.     

The life cycle of Hymenoscyphus fraxineus on Manchurian ash,Fraxinus mandshurica,in Japan

Hymenoscyphus fraxineus causes a lethal disease known as “ash dieback” in the common ash, Fraxinus excelsior, in Europe. It is hypothesized that the fungus originated from East Asia. This fungus is found on the leaf litter of the Manchurian ash, Fraxinus mandshurica, in Japan and is reported to produce apothecia on pseudosclerotial plates formed mainly on decomposing rachises. However, dieback disease has not been reported in Japan, and little is known about the life cycle of H. fraxineus. This study was conducted to explore the behavior and life cycle of this fungus. It was revealed that, after infection by ascospores, H. fraxineus endophytically inhabits the living leaves of F. mandshurica. On fallen leaves, the fungus behaves saprophytically, producing apothecia on pseudosclerotial plates formed mainly on the decomposing rachises. Analysis by real-time quantitative polymerase chain reaction (qPCR) revealed that the amount of H. fraxineus DNA sharply increased in rachises, while such sharp increase of DNA was not found in leaflets.     

Metachromasy of crystal violet in the presence of poly(alpha-L-glutamic acid) and the bound-dye spectra determined by the principal/component-analysis method

In order to study quantitatively the metachromatic behaviour of crystal violet (CV) in the presence of poly (alpha-L-glutamic acid), (poly (Glu)), four sets of the absorption spectra of the poly(Glu)-CV system were analyzed by the extended principal-component-analysis (PCA) method. Two classes of CV-Glu complexes, i.e., the bound-CV species, are present in poly(Glu) regardless of its helical and random-coiled conformations over a wide range of the mixing ratios of Glu residues to CV (P/D). The spectra of the bound CV in a low P/D range < 100 (complex I), extracted by the PCA method, are conformation-dependent showing three absorption bands at 506, ca. 550, and 610-620 nm. The spectra of the bound CV in a high P/D range > 100 (complex II) are closely related to, but not identical with, the free CV. The molar fractions of free CV and complexes I and II, evaluated in the P/D range of 0-150, indicate that CV binds more to the random-coiled poly(Glu) than to the helical one. Metachromasy of CV results from a complicated interplay of an unbound and two differently bound species.     

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.