EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

The selective roles of chaperone systems on over-expression of human-like collagen in recombinant Escherichia coli

Human-like collagen (HLC) is a novel biomedical material with promising applications. Usually, insoluble HLC was formed due to over-expression. In order to improve the production of soluble HLC, the effective chaperone proteins and their mediation roles on HLC were clarified. Trigger factor (TF) pathway with low specificity and high binding affinity to nascent chains could increase soluble HLC expression; GroEL-GroES could increase the expression level of HLC by assisting the correct folding of HLC and increase mRNA level of the gene coding for HLC by enhancing mRNA stability. DnaK chaperone system did not work positively on soluble HLC due to the unbalanced ratio of DnaK:DnaJ:GrpE, especially too high GrpE significantly inhibited DnaK-mediated refolding. The production of soluble HLC with co-expression of exogenous TF and GroEL-GroES was increased by 35.3 % in comparison with the highest value 0.26 g/L reported previously.     

Up-regulation of SKIP relates to retinal ganglion cells apoptosis after optic nerve crush in vivo

Cell cycle re-entry is one of the key processes in neuronal apoptosis. Previous studies have shown that Ski-interacting protein (SKIP) played an important role in cell cycle re-entry. However, its expression and function in optic nerve injury are still with limited acquaintance. To investigate whether SKIP is involved in retinal ganglion cells (RGCs) death, we performed an optic nerve crush (ONC) model in adult rats. Western blot analysis revealed that up-regulation of SKIP was present in retina at 5 days after ONC. Immunofluorescent labeling indicated that up-regulated SKIP was found mainly in RGCs. We also investigated co-localization of SKIP with active-caspase-3 and TUNEL (apoptotic markers) -positive cells in the retina after ONC. In addition, the expression of SKIP was increased in parallel with P53 and P21 in retina after ONC. All these results suggested that up-regulation of SKIP in the retina was associated with RGCs death after ONC.     

Silica nanoparticles induce oxidative stress and inflammation of human peripheral blood mononuclear cells

In the present study, the effects of 10- or 100-nm silica oxide (SiO₂) NPs on human peripheral blood mononuclear cells (PBMC) were examined. Cytotoxic effects and oxidative stress effects, including glutathione (GSH) depletion, the formation of protein radical species, and pro-inflammatory cytokine responses, were measured. PBMC exposed to 10-nm NP concentrations from 50 to 4,000 ppm showed concentration-response increases in cell death; whereas, for 100-nm NPs, PBMC viability was not lost at <500 ppm. Interestingly, 10-nm NPs were more cytotoxic and induced more oxidative stress than 100-nm NPs. Immunoelectron micrographs show the cellular distribution of GSH and NPs. As expected based on the viability data, the 10-nm NPs disturbed cell morphology to a greater extent than did the 100-nm NPs. Antibody to the radical scavenger, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), was used for Western blot analysis of proteins with radicals; more DMPO proteins were found after exposure to 10-nm NPs than 100-nm NPs. Examination of cytokines (TNF-α, IL-1ra, IL-6, IL-8, IL-1β, and IFN-γ) indicated that different ratios of cytokines were expressed and released after exposure to 10- and 100-nm NPs. IL-1β production was enhanced by 10- and 100-nm NPs;, the cytotoxicity of the NPs was associated with an increase in the IL-1β/IL-6 ratio and 100-nm NPs at concentrations that did not induce loss of cell viability enhanced IL-1β and IL-6 to an extent similar to phytohemagglutinin (PHA), a T cell mitogen. In conclusion, our results indicate that SiO₂ NPs trigger a cytokine inflammatory response and induce oxidative stress in vitro, and NPs of the same chemistry, but of different sizes, demonstrate differences in their intracellular distribution and immunomodulatory properties, especially with regard to IL-1β and IL-6 expression.     

Inhibition of deoxyribonuclease activity associated with soybean chloroplasts

The effects of spermidine, pH, ethylene diamine tetracetic acid (EDTA), and adenosine triphosphate (ATP) on deoxyribonuclease (DNase) activity associated with the chloroplasts of soybean (Glycine max (L.) Merr.) were investigated. Chloroplast DNase activity was found to be partially inhibited by either 10 mM spermidine, 20 mM EDTA, or 20 mM ATP. DNase activity was also partially inhibited at non-neutral pH's. Nearly complete inhibition was achieved with use of 30 mM EDTA, pH 10, or a combination of 10 mM spermidine and 10 mM EDTA.     

Polymorphism of DNA Repair Gene xrcc1 and Lung Cancer Risk

The current large-scale meta-analysis was performed to reach a reliable conclusion on the association between X-ray repair cross-complementing 1 (xrcc1) rs1799782 and the development of lung cancer. Studies that investigated the association between rs1799782 and lung cancer risk were identified by searching PubMed. We calculated odds ratio (OR) with corresponding 95 % confidence interval (CI) for Trp/Trp vs Arg/Arg, Trp/Trp + Arg/Trp vs Arg/Arg, and Trp/Trp vs Arg/Trp + Arg/Arg contrast models. Combining all 25 studies, we yielded three summary ORs: 1.07 (95 % CI 0.92–1.23) for Trp/Trp vs Arg/Arg, 0.93 (95 % CI 0.87–1.00) for Trp/Trp + Arg/Trp vs Arg/Arg, and 1.08 (95 % CI 0.94–1.25) for Trp/Trp vs Arg/Trp + Arg/Arg, suggesting rs1799782 was not associated with overall risk of lung cancer. Strikingly, a significantly deceased risk was found among Caucasian populations (Trp/Trp + Arg/Trp vs Arg/Arg, OR = 0.86, 95 % CI 0.76–0.97). This study confirms that xrcc1 rs1799782 may lower the risk of lung cancer among Caucasians.     

The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria

Pseudomonad bacterial are a phylogenetically diverse assemblage of species named within contemporary genera that includePseudomonas, Xanthomonas andAlcaligenes. Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization. A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups. We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning. The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American Type Culture Collection. Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels. Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning. Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case. It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.     

Application of AAO Matrix in Aligned Gold Nanorod Array Substrates for Surface-Enhanced Fluorescence and Raman Scattering

In this paper, we probed surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF) from probe molecule Rhodamine 6G (R6G) on self-standing Au nanorod array substrates made using a combination of anodization and potentiostatic electrodeposition. The initial substrates were embedded within a porous alumina template (AAO). By controlling the thickness of the AAO matrix, SEF and SERS were observed exhibiting an inverse relationship. SERS and SEF showed a non-linear response to the removal of AAO matrix due to an inhomogeneous plasmon activity across the nanorod which was supported by FDTD calculations. We showed that by optimizing the level of AAO thickness, we could obtain either maximized SERS, SEF or simultaneously observe both SERS and SEF together.     

ATPase activities in partially purified membranes of Acetabularia

Partly purified membranes (with plasmalemma material) of Acetabularia mediterranea were studied with respect to ATPase activity in alkali- and Ca⁺⁺-free media and its sensitivity to pH (5 – 9), oligomycin (200 ?g/mg protein), 100 ?M N-N′-dicyclohexylcarbodiimide (DCCD), and 50 ?M vanadate. Besides activities which may originate from mitochondrial H⁺ ATPase (oligomycin-sensitive, alkaline pH optimum) and tonoplast H⁺ ATPase (DCCD-sensitive, pH optimum 7.5), there is ATPase activity with a pH optimum around pH 6.5, sensitive to vanadate and insensitive to DCCD. These results strongly suggest that the electrogenic Cl^? pump in the plasmalemma of Acetabularia is an ATPase. Effects of Mg⁺⁺, Mg-ATP, ADP, GTP, UTP, CTP and HCO₃ ^? versus Cl^? on this ATPase activity are described.     

Modulation of GABA-A receptors of astrocytes and STC-1 cells by taurine structural analogs

Taurine activates and modulates GABA receptors in vivo as well as those expressed in heterologous systems. This study aimed to determine whether the structural analogs of taurine: homotaurine and hypotaurine, have the ability to activate GABA-A receptors that include GABAρ subunits. The expression of GABA-A receptors containing GABAρ has been reported in the STC-1 cells and astrocytes. In both cell types, taurine, homo-, and hypotaurine gated with low efficiency a picrotoxin-sensitive GABA-A receptor. The known bimodal modulatory effect of taurine on GABAρ receptors was not observed; however, differences between the activation and deactivation rates were detected when they were perfused together with GABA. In silico docking simulations suggested that taurine, hypo-, and homotaurine do not form a cation–π interaction such as that generated by GABA in the agonist-binding site of GABAρ. This observation complements the electrophysiological data suggesting that taurine and its analogs act as partial agonists of GABA-A receptors. All the observations above suggest that the structural analogs of taurine are partial agonists of GABA-A receptors that occupy the agonist-binding site, but their structures do not allow the proper interaction with the receptor to fully gate its Cl^? channel.     

Curcumin suppresses proliferation and invasion in non-small cell lung cancer by modulation of MTA1-mediated Wnt/β-catenin pathway

Curcumin, a naturally occurring phenolic compound, has a diversity of antitumor activities. It has been previously demonstrated that curcumin can inhibit the invasion and metastasis of tumors through activation of the tumor suppressor DnaJ-like heat shock protein 40 (HLJ1). However, the specific roles and mechanisms of curcumin in regulating the malignant behaviors of non-small cell lung cancer (NSCLC) cells still remain unclear. In this study, we found that curcumin could inhibit the proliferation and invasion of NSCLC cells and induce G0/G1 phase arrest. Metastasis-associated protein 1 (MTA1) overexpression has been detected in a wide variety of aggressive tumors and plays an important role on cell invasion and metastasis. Our results showed that curcumin could effectively inhibit the MTA1 expression of NSCLC cells. Further research on the subsequent mechanism showed that curcumin inhibited the proliferation and invasion of NSCLC cells through MTA1-mediated inactivation of Wnt/β-catenin pathway. Wnt/β-catenin signaling was reported to play a critical cooperative role on promoting lung tumorigenesis. Thus, these investigations provided novel insights into the mechanisms of curcumin on inhibition of NSCLC cell growth and invasion and showed potential therapeutic strategies for NSCLC.     

On the existence of two GABA pools associated with newly synthesized GABA and with newly taken up GABA in nerve terminals

[¹⁴C]Glutamic acid and [³H]GABA were injected into the lateral ventricle of mouse and then [¹⁴C]GABA and [³H]GABA in synaptosomes isolated from the animals were analysed. The [¹⁴C]GABA was interpreted to be newly synthesized GABA from [¹⁴C]glutamic acid while the [³H]GABA to be newly taken up GABA. We have obtained the following results: (1) when the animals were pretreated with aminooxyacetic acid and thus the GABA content in synaptosomes increased to about 2 times of the control level, only the [³H]GABA was enhanced to 3 times of the control level without any change of [¹⁴C]GABA, (2) the release of [¹⁴C]GABA from synaptosomes by high K⁺ depolarization was 1.5 times greater than that of [³H]GABA, (3) the releases of both [¹⁴C]GABA and [³H]GABA were increased in the presence of cold GABA,l-2,4-diaminobutyric acid or γ-amino-β-hydroxybutyric acid, but only slightly increased in the presence of β-alanine. These results would suggest that newly synthesized GABA and newly taken up GABA localized individually in different pools, which might localize either in different nerve terminals or separately in the same nerve terminal.     

Comparisons of procedures for determining ribosomal ribonucleic acid similarities

Ribosomal ribonucleic acid (rRNA) cistron similarities were determined forClostridium species with labeled preparations of 16S, 23S, or 16S plus 23S rRNA. Similarities were measured by membrane competition and by thermal stability experiments, and the results were correlated. Correlations were also made between 16S rRNA membrane competition homology values and similarities of 16S rRNA oligonucleotides. Very similar results were obtained whether 16S-or 23S-labeled rRNA was used. Clustering patterns were similar for homology values from hybridization experiments incubated at 50° or 60°C, as were the clustering patterns comparing homology values and rRNA hybrid thermal stability values. There was a linear correlation between rRNA homology values and 16S oligonucleotide S_AB values (Tanner et al. [22]) for homology values above 20%, and S_AB values above 0.45.     

The amino-terminal sequence ofStreptomyces griseus carboxypeptidase

The amino-terminal sequence of carboxypeptidase fromStreptomyces griseus was determined using a new protocol for automatic Edman degradation that reduced background noise. The sequence of the first 48 residues is: Asp-Phe-Pro-Pro-Ala-Asp-Ser-Arg-Tyr-His-Asn-Tyr-Ala-Glu-Met-Asn-Ala-Ala-Ile-Asp-Ala-Arg-Ile-Ala-Ala-Asn-Pro-Ser-Ile-Met-Ser-Lys-Arg-Val-Ile-Gly-Lys-Thr-Tyr-Gln-Gly-(Arg)-Asp-Val-Ile-Ala-Val-Lys, which is homologous to that of other zinc-containing carboxypeptidase from vertebrate and invertebrate sources.     

Epigenetic Enhancement of Brain-Derived Neurotrophic Factor Signaling Pathway Improves Cognitive Impairments Induced by Isoflurane Exposure in Aged Rats

Isoflurane-induced cognitive impairments are well documented in animal models; yet, the molecular mechanisms remain largely to be determined. In the present study, 22-month-old male Sprague-Dawley rats received 2 h of 1.5 % isoflurane or 100 % oxygen daily for 3 consecutive days. For the intervention study, the rats were intraperitoneally injected with 1.2 g/kg sodium butyrate 2 h before isoflurane exposure. Our data showed that repeated isoflurane exposure significantly decreased the freezing time to context and the freezing time to tone in the fear conditioning test, which was associated with upregulated histone deacetylase 2, reduced histone acetylation, and increased inflammation and apoptosis in the hippocampus, and impairments of brain-derived neurotrophic factor (BDNF)-tyrosine kinase receptor B (TrkB) and the downstream signaling pathway phospho-calmodulin-dependent protein kinase and phospho-cAMP response element-binding protein. These results suggest that isoflurane-induced cognitive impairments are associated with the declines in chromatin histone acetylation and the resulting downregulation of BDNF-TrkB signaling pathway. Moreover, the cognitive impairments and the signaling deficits can be rescued by histone deacetylase inhibitor sodium butyrate. Therefore, epigenetic enhancement of BDNF-TrkB signaling may be a promising strategy for reversing isoflurane-induced cognitive impairments.     

Ribosomal RNA contents of maize genotypes with different ribosomal RNA gene numbers

Ribosomal RNA (rRNA) contents were determined in 16 maize genotypes whose individual rRNA gene numbers varied from 5000 to 23,000 per 2C nucleus. Analytical polyacrylamide gel electrophoresis of total RNA showed that no obvious relation existed between rRNA gene number and rRNA content. Only two of nine common inbred lines contained more rRNA than W-23, the inbred with the lowest rRNA gene number. Two of four lines with altered protein content (due to long-term experimental selection) had rRNA contents significantly reduced from those of W-23. A line with an apparent duplication of the nucleolus organizer region of chromosome 6 (called 2-NOR) was expected to possess an elevated quantity of rRNA because it possesses a larger nucleolus; however, we produced a 2-NOR isogenic version and found no difference in rRNA content. The rRNA genes in maize are distributed throughout the NOR-heterochromatin and the NOR-secondary constriction portions of the NOR. The absence of an obvious correlation between rRNA gene number and cellular rRNA content may reflect the presence of a large number of rRNA genes in an inactive state, at least during the stage of growth examined in these experiments.     

Modulation,Bioinformatic Screening,and Assessment of Small Molecular Peptides Targeting the Vascular Endothelial Growth Factor Receptor

Vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) are important factors in tumor growth and metastasis. Molecular probes or drugs designed to target VEGF/VEGFR interactions are crucial in tumor molecular imaging and targeted therapy. Bioinformatic methods enable molecular design based on the structure of bio-macromolecules and their interactions. This study was aimed to identify tumor-targeting small-molecule peptides with high affinity for VEGFR using bioinformatics screening. The VEGFR extracellular immunoglobulin-like modules Ig1–Ig3 were used as the target to systematically alter the primary peptide sequence of VEGF_125–136. Molecular docking and surface functional group interaction methods were combined in an in silico screen for polypeptides, which in theory, would have higher affinities for VEGFR. In vitro receptor competition binding assays were used to assess the affinity of the putative VEGFR-binding polypeptides. Rhodamine-conjugated peptides were used to label and visualize peptide-binding sites on A549 cells. Using bioinformatic screening, we identified 20 polypeptides with potentially higher affinity for VEGFR. The polypeptides were capable of inhibiting the binding of ¹²⁵I-VEGF to VEGFR in a dose-dependent manner. The IC₅₀ values of QKRKRKKSRKKH and RKRKRKKSRYIVLS (80 and 185 nmol/L, respectively) were significantly lower than that of VEGF_125–136 (464 nmol/L); thus, the affinity of these peptides for VEGFR was 6- and 2.5-fold higher, respectively, than that of VEGF_125–136. Rhodamine labeling of A549 cells revealed peptide binding mainly on the plasma membrane and in the cytoplasm. Bioinformatic approaches hold promise for the development of molecular imaging probes. Using this approach, we designed two peptides that showed higher affinity toward VEGFR. These polypeptides may be used as molecular probes or drugs targeting VEGFR, which can be utilized in molecular imaging and targeted therapy of certain tumors.