Indomethacin inhibits cell growth of medullary thyroid carcinoma by reducing cell cycle progression into S phase

Indomethacin, a non-steroidal anti-inflammatory drug (NSAID), has been reported to inhibit the growth of medullary thyroid carcinoma (MTC) cells in vitro. However, the mechanism of inhibition of MTC cell growth by indomethacin and its potency have yet to be revealed. We examined the effect of indomethacin on three different MTC cell lines (TT cells, DRO 81-1 cells and HRO 85-1 cells) and two non-MTC cells. The mechanism of indomethacin action in MTC cells was investigated by analyzing intracellular prostaglandin level, apoptosis, and cell cycle in TT cells. Indomethacin inhibited cell growth of all three MTC cell lines but not normal thyroid cells or anaplastic thyroid carcinoma cells. Indomethacin at 10 microM or greater showed a dose response inhibition of cell growth. Indomethacin at 25 muM, a putative therapeutic serum indomethacin level, showed potency similar to 100 to 200 nM sunitinib, a receptor tyrosine kinase inhibitor. To examine whether prostaglandin depletion might determine the inhibition of MTC cell growth, we created different prostaglandin E2 (PGE2) levels in TT cells using three different NSAIDs. A profound PGE2 depletion by indomethacin-ester, a potent cyclooxygenase (COX) II inhibitor, showed the least inhibition of cell growth. Indomethacin did not increase apoptosis of TT cells. Indomethacin, but not naproxen or indomethacin-ester, reduced cell cycle progression into S phase; this was unrelated to the degree of PGE2 depletion. The expression of phosphorylated retinoblastoma (pRb) protein that shifts cells from G(1) to S phase was reduced after exposure to indomethacin. In conclusion, indomethacin has specific anti-tumor effect on MTC cells, probably by reducing cell cycle progression into S phase rather than by prostaglandin depletion. Since no drug therapy is currently available for MTC, indomethacin may be one of the therapeutic candidates.     

Role of 5'-AMP-activated protein kinase in stimulation of glucose transport in response to inhibition of oxidative phosphorylation

Glucose transport is stimulated in a variety of cells and tissues in response to inhibition of oxidative phosphorylation. However, the underlying mechanisms and mediating steps remain largely unknown. In the present study we first tested whether a decrease in the redox state of the cell per se and the resultant increase in generation of reactive oxygen species (ROS) lead to stimulation of glucose transport. Clone 9 cells (expressing the Glut1 isoform of facilitative glucose transporters) were exposed to azide, lactate, and ethanol for 1 h. Although all three agents stimulated glucose transport and increased cell NADH-to-NAD⁺ ratio and phospho-ERK1/2, signifying increased ROS generation, the response to the stimuli was not blocked by N-acetyl-L-cysteine (an agent that counteracts ROS); moreover, the response to azide was not blocked by diamide (an intracellular sulfhydryl oxidizing agent). We then found that cell AMP-to-ATP and ADP-to-ATP ratios were increased and 5'-AMP-activated protein kinase (AMPK) was stimulated by all three agents, as evidenced by increased phosphorylation of AMPK and acetyl-CoA carboxylase. We conclude that although azide, lactate, and ethanol increase NADH-to-NAD⁺ ratios and ROS production, their stimulatory effect on glucose transport is not mediated by increased ROS generation. However, all three agents increased cell AMP-to-ATP ratio and stimulated AMPK, making it likely that the latter pathway plays an important role in the glucose transport response. 5-aminoimidazole-4-carboxamide-1--D-ribofuranoside; extracellular signal related-kinase 1/2; phospho-extracellular signal related-kinase 1/2; N-acetyl-L-cysteine; diamide; acetyl-CoA carboxylase; phospho-acetyl-CoA carboxylase     

Characterization of edible emulsified films with low affinity to water based on kefiran and oleic acid

New edible composite films based on kefiran and oleic acid (OA) at the ratio of 15, 25, and 35% (w/w) were prepared using emulsification with the aim of improving their water vapour barrier and mechanical properties. Film-forming solutions were characterized in terms of rheological properties and particle-size distribution. The impact of the incorporation of OA into the film matrix was studied by investigating the physical, mechanical, and thermal properties of the films. The water vapour permeability (WVP) of the emulsified films was reduced by approximately 33% by adding OA. The mechanical properties of kefiran films were also affected by adding OA: tensile strength was diminished, and elongation increased considerably. Differential scanning calorimetry showed that the glass transition temperature (T_g) of the kefiran film was −16 °C and was not considerably affected by adding OA. Therefore, OA could be incorporated into these films for some food-technology applications that need a low affinity toward water.     

Empirical comparison of cross-platform normalization methods for gene expression data

Background

The prediction of secondary structure, i.e. the set of canonical base pairs between nucleotides, is a first step in developing an understanding of the function of an RNA sequence. The most accurate computational methods predict conserved structures for a set of homologous RNA sequences. These methods usually suffer from high computational complexity. In this paper, TurboFold, a novel and efficient method for secondary structure prediction for multiple RNA sequences, is presented.

Results

TurboFold takes, as input, a set of homologous RNA sequences and outputs estimates of the base pairing probabilities for each sequence. The base pairing probabilities for a sequence are estimated by combining intrinsic information, derived from the sequence itself via the nearest neighbor thermodynamic model, with extrinsic information, derived from the other sequences in the input set. For a given sequence, the extrinsic information is computed by using pairwise-sequence-alignment-based probabilities for co-incidence with each of the other sequences, along with estimated base pairing probabilities, from the previous iteration, for the other sequences. The extrinsic information is introduced as free energy modifications for base pairing in a partition function computation based on the nearest neighbor thermodynamic model. This process yields updated estimates of base pairing probability. The updated base pairing probabilities in turn are used to recompute extrinsic information, resulting in the overall iterative estimation procedure that defines TurboFold. TurboFold is benchmarked on a number of ncRNA datasets and compared against alternative secondary structure prediction methods. The iterative procedure in TurboFold is shown to improve estimates of base pairing probability with each iteration, though only small gains are obtained beyond three iterations. Secondary structures composed of base pairs with estimated probabilities higher than a significance threshold are shown to be more accurate for TurboFold than for alternative methods that estimate base pairing probabilities. TurboFold-MEA, which uses base pairing probabilities from TurboFold in a maximum expected accuracy algorithm for secondary structure prediction, has accuracy comparable to the best performing secondary structure prediction methods. The computational and memory requirements for TurboFold are modest and, in terms of sequence length and number of sequences, scale much more favorably than joint alignment and folding algorithms.

Conclusions

TurboFold is an iterative probabilistic method for predicting secondary structures for multiple RNA sequences that efficiently and accurately combines the information from the comparative analysis between sequences with the thermodynamic folding model. Unlike most other multi-sequence structure prediction methods, TurboFold does not enforce strict commonality of structures and is therefore useful for predicting structures for homologous sequences that have diverged significantly. TurboFold can be downloaded as part of the RNAstructure package at http://rna.urmc.rochester.edu.     

Na,K-ATPase expression in C2C12 cells during myogenesis: Minimal contribution of α2 Isoform to Na,K transport

Summary Cells of the murine skeletal muscle line, C₂C₁₂, undergo differentiation from mononuclear myoblasts to multinuclear myotubes that express a number of proteins associated with striated muscle. We examined the relationship between the abundance of the mRNAs encoding the fast-twitch Ca-ATPase and the α isoforms of Na,K-ATPase and the subsequent expression of their respective polypeptides. Both the mRNA and protein levels of the αl isoform remained constant throughout differentiation. In contrast, the content of mRNAs encoding the α2 isoform and fasttwitch Ca-ATPase increased coordinately with the abundance of their corresponding polypeptides during myotube development. Despite the dramatic increase in α2 expression, estimates of in vitro Na,K-ATPase activity and assessments of in vivo transport activity suggest that α2 contributes little to ionic homeostasis in C₂C₁₂ myotubes.     

Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium

Summary Regulation of Na,K-ATPase mRNA isoform and mRNA expression by thyroid hormone (T₃) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA₁, mRNA₃, and mRNA₁ abundances were nearly constant while mRNA₂ was undetectable. During the interval between postnatal days 5 and 15, mRNA₃ decreased to negligible levels and mRNA₂ became expressed and increased in abundance to account for 20% of the mRNA pool by the 15^th postnatal day. To examine the effect of T₃ on this developmental program, neonates were injected with 75 g T₃/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T₃ treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T₃ treatment, the abundances of mRNA₁, mRNA₃, and mRNA₁ increased, while mRNA₂ continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T₃ augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T₃ does not act as a molecular switch in the developmental expression of the mRNA isoforms in rat myocardium during the first four postnatal days.     

Xenoantisera to human DR antigens: Serological and immunochemical characterization

Antibodies to DR antigens were detected using serological and immunochemical tests in sera from rabbits and goats immunized with cultured human B-lymphoid cells mixed with an anti-T-cell xenoantiserum or with partially purified DR antigens. After absorption with human red blood cells, cultured melanoma cells, and/or T-lymphoid cells, DR xenoantisera become specifically cytotoxic to B lymphocytes. Three out of nine sera tested with a panel of T-depleted peripheral lymphocytes and chronic lymphocytic leukemia cells showed correlation with DR alloantisera submitted to the Seventh International Histocompatibility Workshop. Although the correlation coefficients were lower than those obtained with DR alloantisera, the results obtained suggest that DR xenoantisera may recognize allotypic specificities.     

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis^1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation³. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells^4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis^6,7.Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior⁸. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible⁸.For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.     

Cataclysta lemnata Linnaeus, 1758 (Lepidoptera: Crambidae) expanded its host range feeding on invasive aquatic ferns,species of Azolla Lamarck, 1783 (Hydropteridales: Azollaceae): fitness factors and costs

Cataclysta lemnata Linnaeus, 1758 is a semi-aquatic moth that feeds on Lemnaceae and few of other aquatic plants. In recent years, this moth has been seen feeding on Azolla Lamarck, 1783 Lamarck, J.B. (1783), Encyclopedie methodique: botanique, 1, Paris: Panckouche. 752 pp. [Google Scholar] in Iran in high numbers. The effect of the host plant on preference and performance of the moth was assessed under laboratory conditions. Despite the high survival rate of the young larvae, due to high mortality of the late instars and pupa on Lemna Linnaeus, 1753, the total survival rate on Azolla was higher. Feeding on Azolla increased the development time by almost a week and reduced fertility to half. However, by consuming more food, regulating the egg laying dynamic, and increasing emergence period, the moth retrieved most of the losses and adapted to feed on Azolla. A week before pupation, feeding activity increased between 2 and 8 times in all treatments and on Azolla, the larvae growth rate was slightly higher.     

Interaction of Sulforaphane with DNA and RNA

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO₂), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO₂) interaction. Overall binding constants were estimated to be K_(SFN–DNA)=3.01 (± 0.035)×10⁴ M^-1 and K_(SFN–RNA)= 6.63 (±0.042)×10³ M^-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure.