Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Expression of Glycogen Phosphorylase Isoforms in Cultured Muscle from Patients with McArdle´s Disease Carrying the p.R771PfsX33 PYGM Mutation

Mutations in the PYGM gene encoding skeletal muscle glycogen phosphorylase (GP) cause a metabolic disorder known as McArdle's disease. Previous studies in muscle biopsies and cultured muscle cells from McArdle patients have shown that PYGM mutations abolish GP activity in skeletal muscle, but that the enzyme activity reappears when muscle cells are in culture. The identification of the GP isoenzyme that accounts for this activity remains controversial.

Enhancement of human adipose-derived stem cell expansion and stability for clinical use

Co-culture techniques associating both dermal fibroblasts and epidermal keratinocytes have shown to have better clinical outcome than keratinocyte culture alone for the treatment of severe burns. Since fat grafting has been shown to improve scar remodelling, new techniques such as cell-therapy-assisted surgical reconstruction with isolated and expanded autologous adipose-derived stem cells (ASCs) would be of benefit to increase graft acceptation. Therefore, integrating ASCs into standardized procedures for cultured skin grafting could be of benefit for the patient if cell quality and quantity could be maintained. The purpose of this study was to evaluate ASC processing from adult tissue with simple isolation (without enzymatic steps), expansion (low density of 325-3,000 cells/cm2) and storage conditions to assure methods to enhance the cellular resistance when transferred back to the patient. Co-culture with cell-banked skin progenitor cells (FE002-SK2) showed an increase of 40-50% ASCs yield at high passages alongside with a better preservation of morphology, proper adipogenic and osteogenic differentiation and efficient biocompatibility with 3D collagen scaffolds. ASCs can be considered as a valuable additional cell source to be delivered in biological bandages to the patient in a need of tissue reconstruction such as burn patients.

Synthesis of geranylhydroquinone derivatives with potencial cytotoxic activity

Natural geranylhydroquinone 1 and geranyl-p-methoxyphenol 2 were prepared by Electrophilic Aromatic Substitution (EAS) reactions between geraniol and 1,4-hydroquinone or p-methoxyphenol respectively, using BF3∙Et2O as a catalyst. Furthermore, natural geranylquinone 3, geranyl-1,4-dimethoxyquinone 4 and the new geranyl-4-methoxyphenyl acetate 5 were obtained by chemical transformations of 1 and 2. The compounds were evaluated for their in vitro cytotoxicity activities against cultured human cancer cells of PC-3 human prostate cancer, MCF-7 and MDA-MB-231 breast carcinoma, and Dermal Human Fibroblasts DHF. IC50 values were in the µM range.

Bone Morphogenetic Protein-6 (BMP-6) induces atresia in goat primordial follicles cultured in vitro

This study investigated the effects of bone morphogenetic protein 6 (BMP-6) on in vitro primordial follicle development in goats. Samples of goat ovarian cortex were cultured in vitro for 1 or 7 days in Minimum Essential Medium (control medium) supplemented with different concentrations of BMP-6. Follicle survival, activation and growth were evaluated through histology and transmission electron microscopy (TEM). After 7 days of culture, histological analysis demonstrated that BMP-6 enhanced the percentages of atretic primordial follicles when compared to fresh control (day 0). Nevertheless, BMP-6 increased follicular and oocyte diameter during both culture periods. As the culture period progressed from day 1 to day 7, a significant increase in follicle diameter was observed with 1 or 50ng/ml BMP-6. However, on the contrary to that observed with the control medium TEM revealed that follicles cultured for up to 7 days with 1 or 50ng/ml BMP-6 had evident signs of atresia. In conclusion, this study demonstrated that BMP-6 negatively affects the survival and ultrastructure of goat primordial follicles.

Trisomy 13 mosaicism demonstrated only in skin fibroblasts in a patient presenting psychomotor retardation, pigmentary dysplasia and some dysmorphic features

A Brazilian female infant presented delayed psychomotor development, skin pigmentary dysplasia and some dysmorphic features. Chromosome analysis from peripheral blood culture was normal, but the karyotype from skin fibroblasts revealed mosaicism for trisomy 13. This case demonstrates the relevance of performing chromosomal analysis of skin fibroblasts in patients with mental retardation, associated with pigmentary dysplasia of the skin and a normal karyotype in peripheral blood lymphocytes. To our knowledge, it is the first report of trisomy 13 demonstrated only in skin fibroblasts.

Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ± 0.06% and 2.08 ± 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ± 0.15% and 3.09 ± 0.21% of the islet insulin content in control and 2.43 ± 0.16% and 4.31 ± 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 µg/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets

Interrelationship between adipocytes and fibroblasts during acute damage to the subcutaneous adipose tissue of rats: an ultrastructural study

Ultrastructural phenotypic transitional features were noted between adult adipocytes and fibroblasts in the subcutaneous adipose tissue of the dorsal pad of normal adult Wistar rats of both sexes, weighing 180-260 g, after acute injury either by the implantation of small (1.8 x 1 x 0.4 cm) perforated plastic boxes or by local heat application. Soon after the inflicted damage, fat-containing cells presented variable shapes. Early after damage, some of these cells were round, adipocyte-like, with numerous and large cytoplasmic fat droplets. A few days later, fat-containing cells became elongated, with the fat droplets in their cytoplasm becoming smaller and less numerous. The cells also showed a prominent active rough endoplasmic reticulum and newly formed collagenous matrix accumulated in the interstices. Although current views consider adult adipocytes to be terminal cells, the present findings, in their time sequence, strongly suggest the transformation of adipocytes into fibroblasts after acute injury to adipose tissue.

Effect of dimethylsulfoxide on sphingomyelinase activity and cholesterol metabolism in Niemann-Pick type C fibroblasts

Niemann-Pick type C (NPC) fibroblasts present a large concentration of cholesterol in their cytoplasm due to a still unidentified deficiency in cholesterol metabolism. The influence of dimethylsulfoxide (DMSO) on the amount of intracellular cholesterol was measured in 8 cultures of normal fibroblasts and in 7 fibroblast cultures from NPC patients. DMSO was added to the fibroblast cultures at three different concentrations (1, 2 and 4%, v/v) and the cultures were incubated for 24 h. Sphingomyelinase activity was significantly increased in both groups of cells only when incubated with 2% DMSO (59.4 ± 9.1 and 77.0 ± 9.1 nmol h-1 mg protein-1, controls without and with 2% DMSO, respectively; 47.7 ± 5.2 and 55.8 ± 4.1 nmol h-1 mg protein-1, NPC without and with 2% DMSO, respectively). However, none of the DMSO concentrations used altered the amount of cholesterol in the cytoplasm of NPC cells (0.704 ± 0.049, 0.659 ± 0.041, 0.688 ± 0.063 and 0.733 ± 0.088 mg/mg protein, without DMSO, 1% DMSO, 2% DMSO and 4% DMSO, respectively). This finding suggests that sphingomyelinase deficiency is a secondary defect in NPC and shows that DMSO failed to remove the stored cholesterol. These data do not support the use of DMSO in the treatment of NPC patients.

The requirements of a feasible plastic packaging material for cultured dairy products

The objective of this Master´s Thesis was to conduct a wide scale preliminary survey regarding the package requirements of a cultured dairy package, and to compare the currently used material polystyrene to other suitable packaging materials. Polystyrene has a long history of use in dairy cups, but in recent years its price has increased significantly compared to other common packaging materials. The overall environmental effects of a package and a package material are today a part of designing a sustainable product life cycle. In addition, in certain contexts there has been discussion of the risks posed by styrene polymer for the environment and for humans. These risks are also discussed in this thesis. Polystyrene (PS) is still the most widely used material in dairy cups. In recent years, polypropylene (PP) cups have appeared in increasing numbers on market shelves. This study focuses on the differences of the suitable polymers and examines the suitability of alternative “suitable” polymers with regards to dairy packaging. Aside from focusing on the cup manufacturer, this thesis also examines its subject matter from the viewpoint of the dairy customer, as well as observing the concrete implications of material changes in the overall value chain. It was known in advance that material permeability would be one of the determining factors and that gas transmission testing would be a significant part of the thesis. Mechanical tests were the second part of the testing process, providing information regarding package strength and protectiveness during the package’s life cycle. Production efficiency, along with uninterrupted stable production, was another important factor that was taken into consideration. These two issues are sometimes neglected in similar contexts due to their self-evident nature. In addition, materials used in production may have a surprising significance to the production and efficiency. Consistent high quality is also partly based on material selection. All of the aforementioned factors have been documented and the results have been analyzed by the development team at Coveris Rigid Finland. Coveris is now calculating the total finance effects and capacities should the material changes be implemented in practice. There are many factors in favor of switching to polypropylene at the moment. The overall production costs, as well as the environmental effects of resin production are the primary influences for said switch from the converters’ perspective.

Steps in the formation of neurites and synapses studied in cultured leech neurons

Leech neurons in culture have provided novel insights into the steps in the formation of neurite outgrowth patterns, target recognition and synapse formation. Identified adult neurons from the central nervous system of the leech can be removed individually and plated in culture under well-controlled conditions, where they retain their characteristic physiological properties, grow neurites and form specific chemical or electrical synapses. Different identified neurons develop distinctive outgrowth patterns that depend on their identities and on the molecular composition of the substrate. On native substrates, the patterns displayed by these neurons reproduce characteristics from the adult or the developing neurons. In addition, the substrate may induce selective directed growth between pairs of neurons that normally make contact in the ganglion. Upon contact, pairs of cultured leech neurons form chemical or electrical synapses, or both types depending on the neuronal identities. Anterograde and retrograde signals during membrane contact and synapse formation modify the distribution of synaptic terminals, calcium currents, and responses to 5-hydroxytryptamine.

Bone marrow fibroblasts in patients with advanced lung cancer

In a previous study we demonstrated that the incidence of fibroblast colony-forming units (CFU-F) was very low in bone marrow primary cultures from the majority of untreated advanced non-small lung cancer patients (LCP) compared to normal controls (NC). For this reason, we studied the ability of bone marrow stromal cells to achieve confluence in primary cultures and their proliferative capacity following four continuous subcultures in consecutive untreated LCP and NC. We also evaluated the production of interleukin-1ß (IL-1ß) and prostaglandin E2 (PGE2) by pure fibroblasts. Bone marrow was obtained from 20 LCP and 20 NC. A CFU-F assay was used to investigate the proliferative and confluence capacity. Levels of IL-1ß and PGE2 in conditioned medium (CM) of pure fibroblast cultures were measured with an ELISA kit and RIA kit, respectively. Only fibroblasts from 6/13 (46%) LCP confluent primary cultures had the capacity to proliferate following four subcultures (NC = 100%). Levels of spontaneously released IL-1ß were below 10 pg/ml in the CM of LCP, while NC had a mean value of 1,217 ± 74 pg/ml. In contrast, levels of PGE2 in these CM of LCP were higher (77.5 ± 23.6 pg/ml) compared to NC (18.5 ± 0.9 pg/ml). In conclusion, bone marrow fibroblasts from LCP presented a defective proliferative and confluence capacity, and this deficiency may be associated with the alteration of IL-1ß and PGE2 production.

Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.

Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

Changes in cell shape, cytoskeletal proteins and adhesion sites of cultured cells after extracellular Ca2+ chelation

Although much is known about the molecules involved in extracellular Ca2+ regulation, the relationship of the ion with overall cell morphology is not understood. The objective of the present study was to determine the effect of the Ca2+ chelator EGTA on the major cytoskeleton components, at integrin-containing adhesion sites, and their consequences on cell shape. Control mouse cell line C2C12 has a well-spread morphology with long stress fibers running in many different directions, as detected by fluorescence microscopy using rhodamine-phalloidin. In contrast, cells treated with EGTA (1.75 mM in culture medium) for 24 h became bipolar and showed less stress fibers running in one major direction. The adhesion plaque protein alpha5-integrin was detected by immunofluorescence microscopy at fibrillar adhesion sites in both control and treated cells, whereas a dense labeling was seen only inside treated cells. Microtubules shifted from a radial arrangement in control cells to a longitudinal distribution in EGTA-treated cells, as analyzed by immunofluorescence microscopy. Desmin intermediate filaments were detected by immunofluorescence microscopy in a fragmented network dispersed within the entire cytoplasm in EGTA-treated cells, whereas a dense network was seen in the whole cytoplasm of control cells. The present results suggest that the role of extracellular Ca2+ in the regulation of C2C12 cell shape can be mediated by actin-containing stress fibers and microtubules and by intermediate filament reorganization, which may involve integrin adhesion sites.