Reduction In Cellular Activity Research Articles

NOX1 inhibition attenuates the development of a pro\u2010tumorigenic environment in experimental hepatocellular carcinoma

BackgroundThe poor prognosis of advanced HCC and limited efficacy of current systemic treatments emphasize the need for new or combined targeted therapies. The development of HCC is a multistage process in which liver injury appears in a complex microenvironment associated with oxidative stress. NOX enzymes are the main source of ROS during hepatocarcinogenesis and NOX1 in particular has shown correlation with poor prognosis of HCC patients. This study evaluates the effect of pharmacological NOX1 inhibition on the development and progression of HCC and its effect on the tumor microenvironment.MethodsThe in vitro cytotoxic effects of the NOX1 inhibitor GKT771 (Genkyotex) on human Huh7 and Hep3B and murine Hepa1-6 HCC cell lines, the human THP1 monocyte cell line and mouse macrophages were evaluated via MTT, LDH activity and CaspGlo® assays. In order to induce in vivo HCC, male SV129 wild-type mice received weekly IP injections of diethylnitrosamine (DEN) (35 mg/kg) for 20–25 weeks. Mice were treated with vehicle or GKT771 (30 mg/kg) via oral gavage, daily or twice daily, in preventive and therapeutic studies. The liver damage was evaluated for inflammation, angiogenesis, fibrosis and HCC development via histology, RT-qPCR, multiplex analyses and ROS levels.ResultsA concentration-dependent reduction in cellular activity of the human HCC cell lines without cytotoxicity was observed. GKT771 treatment reduced LPS-induced pro-inflammatory bone-marrow derived macrophage polarization. DEN injections resulted in 100 % tumor formation and the induction of HCC markers which could be reduced by twice daily dosing of GKT771 at early onset of advanced HCC. DEN-induced HCC resulted in an upregulation of pro-inflammatory, angiogenic and fibrotic markers which was less pronounced in GKT771 treated mice in all treatment regimens. In line, liver fibrosis was induced in HCC mice and this to a lesser extend upon GKT771 treatment.ConclusionsNOX1 inhibition showed to be safe and well tolerated and was able to attenuate the induction of a pro-inflammatory, angiogenic and pro-fibrotic microenvironment suggesting that this might be a promising adjuvant therapeutic strategy in the treatment of advanced HCC.

Self-Incompatibility Triggers Irreversible Oxidative Modification of Proteins in Incompatible Pollen.

Self-incompatibility (SI) is used by many angiosperms to prevent self-fertilization and inbreeding. In common poppy (Papaver rhoeas), interaction of cognate pollen and pistil S-determinants triggers programmed cell death (PCD) of incompatible pollen. We previously identified that reactive oxygen species (ROS) signal to SI-PCD. ROS-induced oxidative posttranslational modifications (oxPTMs) can regulate protein structure and function. Here, we have identified and mapped oxPTMs triggered by SI in incompatible pollen. Notably, SI-induced pollen had numerous irreversible oxidative modifications, while untreated pollen had virtually none. Our data provide a valuable analysis of the protein targets of ROS in the context of SI-induction and comprise a benchmark because currently there are few reports of irreversible oxPTMs in plants. Strikingly, cytoskeletal proteins and enzymes involved in energy metabolism are a prominent target of ROS. Oxidative modifications to a phosphomimic form of a pyrophosphatase result in a reduction of its activity. Therefore, our results demonstrate irreversible oxidation of pollen proteins during SI and provide evidence that this modification can affect protein function. We suggest that this reduction in cellular activity could lead to PCD.

A biocompatible decellularized pulp scaffold for regenerative endodontics.

To investigate the feasibility of decellularizing the entire dental pulp using a mild treatment protocol to develop a decellularized biological extracellular matrix scaffold for use in regenerative endodontic procedures. Decellularized human dental pulps were assessed using histological and immunohistochemical methods, scanning electron microscope and DNA quantification assay. Cytotoxicity assays to determine decellularized scaffold biocompatibility were also performed. Decellularized scaffolds were seeded with human dental pulp stem cells and cell viability assessed using Live/Dead® stain. Quantitative data were analysed statistically using Student's t-test and one-way analysis of variance to compare mean values between groups depending on group numbers. Assessment of decellularized tissues revealed an acellular matrix with preservation of native tissue histoarchitecture and composition. Decellularized tissues showed no evidence of cytotoxicity, with cell growth in direct contact with the scaffold and no reduction in cellular activity following extract incubation. Furthermore, the scaffold was able to support human dental pulp stem cell viability and attachment following recellularization. Promising results were observed in developing a decellularized biological scaffold derived from the dental pulp with the perseveration of extracellular structural components which are required for tissue-specific regeneration.

In vitro osteocytic microdamage and viability quantification using a microloading platform

Bone remodeling is a process in which bone is resorbed by osteoclasts and formed by osteoblasts. This is normally a paired process, although it can be disrupted by changes in mechanical load. One theory is that osteocytes play a key role in the cellular regulation of this process. Mechanotransduction studies, which investigate how cells convert mechanical stimuli into biophysical effects and cellular activity, offer one way to investigate this theory. Mechanotransduction work is commonly done by applying an isolated mechanical load to cells grown in vitro, and quantifying the response. While in vitro work does not fully replicate the natural environment, it does allow the study of isolated factors. In this study, a mechanical loading platform was designed, fabricated, and characterized for bone mechanotransduction studies. This platform was designed to tent cell-seeded substrates from below, loading using out of plane distension. This introduced a nonuniform strain profile, enabling the study of cells cultured under identical conditions and variable strains as a function of substrate location. An alphanumerically gridded polydimethylsiloxane well substrate was designed and fabricated for cellular loading experiments. Following initial characterization, a study was run to quantify the cellular activity of osteocyte-like MLO-Y4 cells as a function of strain field. The results indicated that regions with lower strains led to an increase in cellular activity while higher strains led to a reduction in cellular activity. This demonstrated that cells could be exposed to mechanically-induced microdamage using the microloading platform.

Coculture of Notochordal Cells with Nucleus Pulposus and Annulus Fibrosus Cells under Normoxia and Hypoxia

Introduction Notochordal cells (NC) are shifted back into focus due to their apparent action of activating other disc cells via indirect release of yet unknown factors into the medium (conditioned medium = CM).1,2 Recent evidence confirms the results from the late 1990s.3,4 Here, we test porcine (p) NC cultured in 3D and the influence of adding serum or using serum-free medium onto the culture on NC cells and its stimulating effects for subsequent coculture with primary bovine (b) nucleus pulposus (bNPC) and annulus fibrous cells (bAFC). Materials and Methods Primary pNC, bNPC, and bAFC were isolated from porcine tails (< 6-12 months age) or bovine tails (∼1 year age), which were obtained from the food chain (N = 4 repeats) within 4 hours postmortem. All cells were seeded into 1.2% alginate, each with a density of 4 × 106/mL. NC were then either cultured for 7 days in serum free medium (SFM = Dulbecco modified eagle medium [DMEM] supplied with ITS+, 50 µg/mL vitamin C and nonessential amino acids) or DMEM + 10% fetal calf serum (FCS). CM was produced from NC collecting 4 mL SFM and keeping approximately 30 beads for 7 days. Then, a coculture was set up in SFM for 14 days using indirect cell-cell contact (culture insert, high density pore, 0.4 µm) using a 50:50% ratio5 of pNC:bNP or bAF, or by addition of CM, respectively. The cell activity, glycosaminoglycan per DNA (GAG/DNA) ratio, and real-time RT-PCR of IVD relevant genes were monitored. Mass spectrometry was performed on the SFM and the cocultured medium as well as the CM of the pNC to identify possible key cytokines to the stimulatory effects. Results The results for cell activity confirmed that pNC are highly responsive on the nutritional condition in the culture (K-W test, p = 0.048) after 7 days of coculture. bNPC and bAFC did not respond significantly different to coculture or addition of CM with respect to cell activity. However, GAG/DNA ratio of pNC was significantly upregulated if they were initially pre-exposed to FCS and in coculture with bNPC after 14 days, for both normoxia and hypoxia (K-W, p = 0.03). The bNPC were stimulated by both, 1:1 coculture with pNC but also by addition of CM only, which resulted in approximately 200% increased GAG/DNA values relative to the day 0 state. However, this doubling of the GAG/DNA ratio was nonsignificant after 14 days. The aggrecan/collagen type 2 ratio as quantified from real-time RT-PCR pointed to a beneficial state of the bNPC if cultured either in indirect coculture with pNC or by the addition of CM (Fig. 1). The mass spectrometric analysis of the CM revealed that there was connecting tissue growth factor present (CTGF) among the cytokine CLC11, a cytokine that has been found to be expressed in skeletal tissues including bone marrow and chondrocytes among other factors that might have immunoregulatory and cell proliferative functions.

The Neurotoxic Eff ect of Intrathecal Diclofenac Sodium in Rats

Summary We aimed to investigate the possible neurotoxic eff ects of single and repeated doses of diclofenac sodium administered to rats. The current study included 24 male Sprague-Dawley rats weighing 250-300 g. At the end of a 4-h fasting period, the rats were randomly split into 3 groups following the establishment of anesthesia with intraperitoneal delivery of 100 mg kg -1 of ketamine hydrochloride and 10 mg kg -1 of xylazine chloride. Group 2 (n=8) received 10 μl (200 μg) of intrathecal diclofenac sodium on 7th day as a single dose, whereas the rats in group 1 (n=8) and group 3 (n=8) were received 10 μl of intrathecal 0.9% saline and 10 μl (200 μg) of intrathecal diclofenac sodium via a 0.40 x 50 mm needle per day for 7 days. During the course of the study the animals were examined with regard to clinical toxicity. After fixating the tissues by injection of 10% formaldehyde, spinal cords were explored and evaluated histopathologically with electron microscopy. Statistical analysis was performed with the Kruskal Wallis test. P values <0.05 were considered statistically significant. While electron microscopic examination showed no changes in the control group, diclofenac sodium exhibited neurotoxic eff ects that were more marked following the 7-days treatment. Diclofenac sodium was neurodegenerative, depending on the dose, and cellular organelles were observed to have compression associated with extracellular edema. Neurodegeneration was thought to be occured with a significant reduction in cellular activity.

Cytotoxicity and Apoptotic Effect of Phytoceramide IV Containing Liposomes on Murine Mastocytoma Cell P815

The cytotoxicity and apoptotic effect of phytoceramide IV ( N-palmitoyl-phytospingosine) were investigated by preparaing the ceramide containing liposomes using cholesteryl hemisuccinate (CHEMS) and methoxypolyethylene glycol (2000) cholesteryl succinate (PEGCHS). The ceramide-containing liposomes prepared using ultrasonication contained CHEMS and the phytoceramide IV at a molar ratio of 1:1. Gel chromatography and high performance liquid chromatography equipped with an evaporative light-scattering detection (HPLC-ELSD) were used to analyze the liposomes. The results show that the ceramide entrapment efficiency is over 90% and the molar ratio of phytoceramide IV to CHEMS is 1:1.4. The ratio of ceramide to CHEMS as well as the ultrasonication duration affects the liposome properties. The phytoceramide IV encapsulated in the liposomes reduces the cellular activity of the murine mastocytoma cell line P815 in a dose-dependent manner and the reduction of cellular activity is due to cell apoptosis. 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) staining further supports this result. The encapsulation of highly hydrophobic ceramides into liposomal formulations could become a potential candidate for enhanced apoptosis.

Tooth morphology in Down's syndrome: evidence for retardation in growth.

Two dental morphological features, lower incisor shape and Carabelli trait on upper molars, were studied in Down's syndrome (DS) individuals and control groups. Lower incisors were smaller in DS in absolute terms, but showed a relatively greater mesiodistal crown dimension than labiolingual, the opposite relationship to normal. Although the Carabelli trait was present in a high percentage of DS subjects, few showed the cuspal form. These findings are consistent with the concept of a reduction of cellular activity of tooth germs reflecting a general retardation in growth in DS. The altered shape of the lower incisors may influence the pattern of periodontal disease observed in DS subjects.

Mucosus Organism from Suppurative Lesions of Rat on Diet Deficient in Vitamin A

Spontaneous infection of the upper part of the respiratory tract of the albino rat is not uncommon. In animals being used for experimental purposes, as well as among those kept in stock 1 one frequently encounters suppurative otitis media, purulent nasal sinusitis, and various types of pulmonary infections. As terminal events in the course of experimental avitaminosis in the white rat, such infections are frequent 2 and are instrumental in causing death. It is thought that deficiency of vitamin A causes such a reduction in cellular activity that it amounts to a deficiency of glandular activity 3 and that this, in turn, brings about secondary changes, bacterial invasion, and death. Reviewing the literature, one is surprised to find that bacteriologic studies of these infections have not been extensive. For this reason, and because it was desired to find an organism in rats kept on diets deficient in vitamin A that might be suitable for use in the study of the relationship of vitamin deficiencies to infection, cultural studies of a group of animals dying from avitaminosis, were made. Method of Culture.-By using ordinary skill one can obtain satisfactory cultures from the middle ears and the nasal turbinates of a rat by the following technique.